In recent years, the bacteriophage Φ29 (Phi29) DNA polymerase has garnered increasing attention due to its high-fidelity amplification capacity at constant temperatures. To advance the industrial application of this type of isothermal polymerases, this study mined and characterized new enzymes from the microbial metagenome based on the known Phi29 DNA polymerase sequence. The results revealed that a new enzyme, Php29 DNA polymerase, was identified in the microbial metagenome with plants as the hosts. This enzyme exhibited higher strand displacement activity, with a 59.5% similarity to bacteriophage Φ29. Experimental validation demonstrated that the enzyme had 3'→5' exonuclease activity, and its amplification products can serve as substrates for further catalytic reactions. The discovery and validation of Php29 DNA polymerase gives insights into the future industrial application of isothermal polymerases.
DNA-Directed DNA Polymerase/metabolism* ; Bacillus Phages/genetics* ; Metagenome

Objective:To investigate the predictive value of N-terminal pro-brain natriuretic peptide (NT-proBNP), cystatin C (CysC) and interleukin-17 (IL-17) in relapse after radiofrequency catheter ablation (RFCA) in elderly patients with persistent atrial fibrillation.Methods:The clinical data of 69 elderly patients with persistent atrial fibrillation underwent RFCA (atrial fibrillation group) in Shanghai Pudong New Area People′s Hospital from January 2020 to December 2021 were retrospectively analyzed. Additionally, 69 healthy subjects underwent physical examinations during the same period were selected as the healthy control group. The levels of serum NT-proBNP, CysC and IL-17 were detected. The relapse after RFCA was recorded. Multivariate Logistic regression was used to analyze the independent risk factors of relapse after RFCA in elderly patients with persistent atrial fibrillation. The values of NT-proBNP, CysC and IL-17 in predicting the relapse after RFCA in elderly patients with persistent atrial fibrillation were evaluated by the receiver operating characteristics (ROC) curve.Results:The serum NT-proBNP, CysC and IL-17 before operation and 7 d after operation in atrial fibrillation group were significantly higher than those in healthy control group: (789.41 ± 89.22) and (358.96 ± 50.24) ng/L vs. (114.38 ± 32.56) ng/L, (1.42 ± 0.30) and (1.20 ± 0.21) mg/L vs. (0.98 ± 0.17) mg/L, (12.48 ± 3.21) and (9.83 ± 2.58) ng/L vs. (7.85 ± 2.13) ng/L, and there were statistical differences ( P<0.05); compared with healthy control group, there were no statistical difference in the indexes 1 and 3 months after operation ( P>0.05). The serum NT-proBNP, CysC and IL-17 7 d, and 1, 3 month after operation in atrial fibrillation group were significantly lower than those before operation, and there were statistical differences ( P<0.05). The 69 patients were followed up for 1 year, with 20 cases experiencing relapse and 49 cases not experiencing relapse. There were no statistical differences in the serum NT-proBNP, CysC and IL-17 before operation and 7 d after operation between relapse patients and non-relapse patients ( P>0.05); the serum NT-proBNP, CysC and IL-17 1 and 3 months after operation in relapse patients were significantly higher than those in non-relapse patients, 1 month after opertion: (132.49 ± 32.84) ng/L vs. (115.56 ± 27.61) ng/L, (1.10 ± 0.15) mg/L vs. (0.99 ± 0.12) mg/L and (8.59 ± 1.76) ng/L vs. (7.65 ± 1.58) ng/L; 3 months after operation: (140.37 ± 32.83) ng/L vs. (119.90 ± 25.44) ng/L, (1.17 ± 0.20) mg/L vs. (1.02 ± 0.15) mg/L and (9.12 ± 2.31) ng/L vs. (7.74 ± 1.80) ng/L, and there were statistical differences ( P<0.05 or<0.01). Multivariate Logistic regression analysis result showed that the serum NT-proBNP, CysC and IL-17 1 and 3 months after operation were the independent risk factors of relapse after RFCA in elderly patients with persistent atrial fibrillation ( P<0.01). ROC curve analysis result showed that the area under curve (AUC) of the serum NT-proBNP, CysC, IL-17 and the combination of three indexes 3 months after operation in predicting the relapse after RFCA in elderly patients with persistent atrial fibrillation were higher than those at 1 month after operation (0.813 vs. 0.783, 0.770 vs. 0.721, 0.725 vs. 0.717 and 0.927 vs. 0.833; P<0.05), the AUC of combination of three indexes 1 and 3 months after operation was significantly higher than that of individual indexes at each time point ( P<0.05). Conclusions:The elevated levels of serum NT-proBNP, CysC and IL-17 after operation in elderly patients with persistent atrial fibrillation are closely related to the relapse after RFCA, and can be used as biochemical indicators to predict recurrence.

Objective To investigate the mutations of epigenetic regulation factor ASXL1 gene in myelodysplastic syndrome(MDS).Methods Mutation analysis of ASXL1 gene in 53 de novo MDS patients and 20 healthy persons was performed by using polymerase chain reaction(PCR)followed by sequence analysis at DNA level.The clinical and laboratory characteristics were compared in MDS patients with ASXL1 gene mutation and ASXL1 wild type.ASXL1 mutation in mRNA level was detected by using reverse transcription PCR(RT-PCR)followed by sequence analysis.Results ASXL1 gene mutations were observed in 9 cases(16.9%)of 53 MDS patients.6 mutation types were detected,including 4 frameshift mutations types(2 cases with p.Glu635ArgfsX15,3 cases with p.Gly646TrpfsX12,1 case with p.Ala640GlyfsX14 and 1 case with p.Gly790TrpfsX10)and 2 nonsense mutation types(1 case with p.Gln1063X and 1 case with p.Gln695X).All the mutations were heterozygous,and p.Gly790TrpfsX10 and p.Gln695X were new mutation types.In addition,a single nucletide polymorphism(SNP)p.Gly652Ser was also detected in 4 cases with MDS.5 cases of p.G652S SNP and 1 case of p.Leu1173Leu SNP were detected in 20 healthy people.Frameshift mutation(p.Gly646TrpfsX12)could be detected at mRNA level by using RT-PCR.Differences were not observed in red blood cell counts,white blood cell counts,platelet counts,hemoglobin levels,reticulocyte,neutrophil granulocyte,the peripheral blood lymphocytes percentage,T-cell subsets in the peripheral blood,the proportion of primitive cell in the marrow and MDS types between the patients with ASXL1 gene mutation and ASXL1 wild type patients(P >0.05).Conclusion There is a high frequency of ASXL1 gene mutation in MDS patients,which can be detected at mRNA level.

This study was aimed to reveal the material basis on different diseases of the same syndrome damp-heat syndrome from the level of metabonomics.The typical damp-heat syndrome patients diagnosed as chronic viral hepatitis B,non-alcoholic fatty liver disease,or chronic glomerulonephritis were included,with 30 cases in each disease.There were 30 healthy volunteers in the control group.The serum samples were detected by UPLC-QTOFMS and GC-TOFMS.And then,the results were analyzed by variance analysis in order to find out the generality and specificity of metabolic material in three different diseases with damp-heat syndrome.The results showed that through comparisons of different diseases with damp-heat syndrome,as well as the healthy group as control,it was revealed that inosine,uridine,aspartic acid,oleic acid glyceride and lactate were the same substances of three diseases of damp-heat syndrome.It was concluded that based on metabonomics,as for three different diseases with damp-heat syndrome,there were different substances among different diseases,but common substances related to damp-heat syndrome.Thus,it provided objective evidences for the theory of different diseases of the same syndrome in traditional Chinese medicine (TCM) from the level of metabonomics.

Objective: To investigate the effect of biology and mTOR pathway activity of down-regulated TSC2 gene expression on U937 leukemia cells. Methods: Gene expression was down-regulated by lentivirus induced RNA interference on TSC2 high expressed U937 cell line; the proliferation, apoptosis and differentiation were detected by CCK-8 assay, colony formation assay and flow cytometry; the gene expression level and protein kinase activity were detected by qRT-PCR and Western blot. Results: Down-regulated expression of TSC2 gene promoted U937 cell proliferation and colony formation ability (P<0.05) . The proportion in G₀/G₁ phase of TSC2 down-regulated U937 cell was much lower than that of the control cells [ (52.53±3.75) % vs (75.10±4.33) %, t=6.829, P=0.002], the S phase [ (22.43±1.00) % vs (15.47±1.20) %, t=-5.581, P=0.019] and G₂/M phase [ (25.03±4.34) % vs (14.33±0.91) %, t=-5.413, P=0.013] was remarkably higher than that of the control cells (P<0.05) . There were no statistically significant differences in cell apoptosis and differentiation (P>0.05) . Down-regulation of TSC2 led to the increased activity of mTOR, 4EBP1 and S6K1, but did not influence the activity of AKT. The expressions of proliferation related cyclinD1, c-myc and PTEN were also up-regulated after TSC2 silenced, but the expressions of P27KIP and BCL-XL were not changed. Conclusion Downregulation of TSC2 could promote the proliferation of U937 cells through up-regulation of mTOR activity.

Objective To explore the value of the plasma miR-193a-5p level on diagnosis and monitoring the response to treatment in acute myeloid leukemia (AML). Methods Peripheral blood samples were obtained from AML patients enrolled in hematology department of the Second Hospital of Shanxi Medical University from July 2015 to December 2015, including 30 de novo AML patients, 9 patients in complete remission (CR) and 6 patients in relapse. Peripheral blood samples from 15 healthy people were randomly choosed as the health control group. Plasma miR-193a-5p expression levels were detected by using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Results The plasma miR-193a-5p relative expression level of AML patients group [0.465 6 (0.103 1-5.000 2)] was obviously lower than that of health control group [0.766 1 (0.052 1-3.134 4)] (U= 122, P= 0.018 7). The plasma miR-193a-5p relative expression levels of de novo group and relapse AML group were significantly lower than those of CR group and health control group (P<0.05), and there was no significant difference between the CR group and health control group (U= 56, P= 0.511 9). No significant correlation was found between the plasma miR-193a-5p level and age, gender, blast percentage of the bone marrow, peripheral blood leukocyte count, platelet count, CD34+cells'percentage and so on. Conclusion The decreased plasma miR-193a-5p expression level can be served as a new and noninvasive biomarker for the evaluation of diagnosis and treatment in AML.

OBJECTIVETo identify the MPL L391-V392ins12 spliceosome and analyze its frequencies in patients with myeloproliferative neoplasms (MPN).

METHODSMPL aberrant spliceosome was identified through reverse transcription polymerase chain reaction (RT-PCR)combined with cloning sequencing. The mutation of this spliceosome in 248 MPN patients and 200 normal people was determined by allele-specific polymerase chain reaction (AS-PCR).

RESULTSA novel aberrant spliceosome of MPL gene (MPL L391-V392ins12)was identified, i.e. 36 bp intron was retained between exon7 and exon8, and there were 12 amino acids (EGLKLLPADIPV)inserted. MPL L391-V392ins12 mutation was detected in 19 (7.66%)of the 248 patients with MPN, including 1 (1.92%) of 52 patients with PV, 14 (9.66%) of 145 with ET, and 4 (7.84%) of 51 with PMF. And the mutation was not detected in the group of 200 normal people.

CONCLUSIONMPL L391-V392ins12 spliceosome is an aberrant spliceosome present in the MPN. It can be detected in PV, ET and PMF, and more frequently in ET and PMF. This mutation may play an important role in the process of MPN.

Humans ; Mutation ; Myeloproliferative Disorders ; genetics ; Neoplasms ; genetics ; Polymerase Chain Reaction ; Receptors, Thrombopoietin ; genetics ; Spliceosomes

Objective: To investigate the effect of losartan on angiotensin II (Ang II) expression and myocardial remodeling in myocardial infarction (MI) rats’ model.
Methods: A total of 32 SD male rats were divided into 4 groups, Sham operation group, MI group, MI with losartan 10mg/(kg·d) group and MI with losartan 20mg/(kg·d). n=8 in each group. MI model was established and the electrocardiogram changes before and after MI were recorded, hemodynamic indexes were detected at 4 weeks after MI, pathological changes of myocardial tissue were examined by HE staining. The myocardial mRNA and protein expressions of ACE2 and Ang II were detected by RT-PCR and Western Blot analysis.
Results: Compared with Sham operation group, MI group showed increased LVMI and decreased LVEF P<0.05;the above changes were getting better in both MI with losartan groups in a dose-dependent manner. The pathological examination presented that MI group had myocardial cell swelling, fracture, hyperplasia and inflammatory cell infiltration, those damages were less in MI with losartan groups in a dose-dependent manner, Sham operation group had no pathological changes. Compared with Sham operation group, the mRNA and protein expressions of Ang II were obviously higher in MI group, P<0.05 and the expressions were decreased in MI with losartan groups in a dose-dependent manner;the mRNA and protein expressions of ACE2 were slightly increased in MI group and the expressions were further increased in MI with losartan groups in a dose-dependent manner.
Conclusion: Losartan could increase ACE2 expression and therefore, inhibit Ang II expression and improve the ventricular remodeling in MI rats’ model.

Objective To investigate the diagnostic values of direct lymphangiography for the thoracic duct outlet obstruction.Methods The image data of direct lymphangiography were retrospectively analyzed in 124 patients with lymphedema,Chylothorax,chylous ascites,chyluria and intestinal lymphangiectasis,and compared with the results of neck thoracic duct surgical exploration,2 radiologists reviewed DLG DSA images in a double blind manner.The number of neck stem,subclavian stem,bronchialmediastinal stem and TD terminal into blood obstruction on the operation side showed by DLG were assessed using Kappa analysis.Results Of 124 patients,80 patients had the left cervical lymphatic stem reflux on DLG,75 patients with the left subclavian lymphatic stem reflux,30 patients with the left bronchial-mediastinal lymphatie stem reflux,118 patients showed the thoracic duct outlet barrier into the blood.The consistency rate of DLG were 89.9％ (80/89),92.6％ (75/81),90.9％ (30/33) and 95.2％ (118/124) compared with the neck thoracic duct surgical exploration.Tow radiologists had a high degree of diagnostic consistency (K =0.82,P ＜ 0.05).In addition,114 patients (91.9％) had tortuous,dilated waist lymphatic stem,only 10 patients (8.1％) were normal.The cisterna chyli reflux were found in 92 patients (74.2％),intestinal stem reflux in 16 patients (12.9％),reflux to the kidney area in 11 patients (8.9％),to the pericardium reflux in 5 patients (4.0％),vaginal lymphatic leakage in 7 patients (5.6％),retroperitoneal lymph leakage in 2 patients (1.6％),pleural lymphatic leakage in 3 patients (2.4％),tracheal lymph leakage in 1 patient (0.8％).Conclusion Direct lymphangiography has a high consistency with the cervical thoracic duct surgical exploration in displaying thoracic duct outlet obstruction.