Objective To determine the effects of hypoxia inducible factor-1α(HIF-1α) expression on postoperative adjuvant radiotherapy in esophageal carcinoma. Methods 95 cases with esophageal carcinoms who received radical operation were analyzed with followed-up data from 1995 to 1998.Expression of HIF-1α in 45 patients with esophageal carcinoma who received radiotherapy after radical operation were deternlined by immunohistochemical method in contrast with 50 patients with esophageal carcinoma received surgery alone.Kaplan-Meier method and COX proportional hazard model were used to analyze.Results The positive expression of HIF-1α in esophageal carcinoma was observed mainly in the nucleus of tumor cells.The positive expression rate of HIF-1α in esophageal carcinoma was 58.9%.The expression of HIF-1α had no relationship with age,sex,histologic subtype and T stage,but had positive relationship with recurrence and distant metastasis.There was significant difference between patients with positive and negative HIF-1α protein expression in surgery alone and postoperation radiotherapy group. COX model analysis showed that HIF-1α had separate and significant impacts on prognosis in surgery group and surgery plus radiotherapy group. Conclusion Over expression of HIF-1α protein suggests a poor prognosis,and has tendency to resist radiotherapy in esophageal carcinoma.

Abstract: Objective　To genotype and analyze whole genomic features of Coxsackievirus B3 (CVB3) isolated in Tianjin, to improve evolution information of CVB3 virus in Tianjin, and to provide basis for surveillance and early warning of related diseases. Methods　Viral RNA was extracted from five CVB3 strains isolated in Tianjin, whole genome sequence of the virus was amplified by RT-PCR and sequenced by next-generation sequencing method, and phylogenetic and recombinant analysis were carried out. Results　The open reading frame 1(ORF) of the five CVB3 strains contained 6 555 nucleotides and encoded 2 185 amino acids, and ORF2 was composed of sequences encoding 68 amino acids. The nucleotide sequence similarity ranged from 78.3%-100%, and the amino acid sequence similarity ranged from 95.7%-100%. Compared with the CVB3 prototype strain, the nucleotide sequence similarity of the five viruses was between 78.2%-79.1%, and the similarity of amino acid sequences was 94.9%-95.3%. All five viruses exhibited a T151A mutation on the VP2 protein. Additionally, the encephalitis isolate showed a K158E mutation on the VP2 protein, while one of the sewage isolates had a C234T mutation in 5' noncoding region. The five strains belonged to two different genotypes, among which the encephalitis isolate in 2016 belonged to the D genotype, while the sewage isolates in 2021 belonged to the E genotype. This is also the first report of E genotype CVB3 in northern China. The CVB3 strain may have recombinant events in non-structural protein regions, in which encephalitis isolate may recombine with a Coxsackievirus B5 (CVB5) strain, while sewage isolates may have recombinant events with a strain of ECHO virus 18 (E18). Conclusions　The CVB3 isolates in Tianjin belong to D and E genotypes, and recombination events may exist in non-structural protein region of the viral genome. The results of CVB3 virus genome analysis in sewage suggests presence of CVB3 infection in the population of Tianjin, and its epidemic dominant genotype may have changed.

Objective To compare the degree of cell injury induced by 3D protein (SDLY11 and SDLY107) of enterovirus 71 (EV71) strains.Methods EV71 strains SDLY11 and SDLY107 were respectively isolated from children with mild and severe hand foot mouth disease.The target genes 11-3D-Flag and 107-3D-Flag were amplified by reverse transcription-polymerase chain reation (RT-PCR) and inserted into the eukaryotic expression vector pcDNA3.1.The recombinant plasmids 11-3D-Flag-pcDNA3.1 and 107-3D-Flag-pcDNA3.1 were transformed into Escherichia.coli DH5α, respectively, and were identified by enzyme digestion and sequencing.The recombinant plasmids were transfected into rhabdomyosarcoma (RD) cells, respectively.Expression of 3D protein was detected by indirect immunofluorescence assay and western blot.Cell injury induced by 3D protein was detected with lactate dehydrogenase (LDH) test, cell proliferation was detected by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenylthiazolium bromide (MTT) test, and cell apoptosis was detected with Annexin-V and PI.Multiple comparisons among groups were analyzed using LSD-t test if multiple sets of variables were consistent with homogeneity of variance.If not, Dunnett T3 test was used.Results The 1 400 bp fragments were amplified by reverse tramscription (RT)-polymerase chain reaction (PCR), and the recombinant plasmids were digested by enzyme and the 1 400 bp and 5 400 bp fragments were obtained and identified.Gene sequencing showed that the sequences were consistent with the target genes.The specific fluorescence was observed by indirect immunofluorescence assay, and the western blot showed that the molecular weight of the target protein was 55×103.The LDH test showed that the A490 of SDLY11 3D protein transfection group (0.790±0.048) was higher than that of SDLY107 3D protein transfection group (0.641±0.018).The difference was statistically significant (t=5.14, P<0.05).The cell membrane damage caused by SDLY11 3D protein was more severe than SDLY107 3D protein.The MTT test showed that the A570 of SDLY11 3D protein transfection group (1.028±0.020) was lower than that of SDLY107 3D protein transfection group (1.081±0.002), and the difference was statistically significant (t=3.31, P<0.05).The effect on cell proliferation activity of SDLY11 3D protein was greater than SDLY107 3D protein.The results of Annexin-V/PI showed that the percentage of apoptotic cells of SDLY11 3D protein transfection group and SDLY107 3D protein transfection group were (1.471±0.246)% and (1.465±0.237)%, respectively, and the difference was not statistically significant (t=0.04, P=0.973).Conclusions Compared with the SDLY11 3D protein, SDLY107 3D protein induces slighter cell injury, has weaker effect on cell proliferation activity, and is more favorable for virus replication in cells.

Objective To investigate the surgical technique and outcomes of replacement of chordae tendineae in mitral valve repair, and evaluate the value of real-time three-dimensional transesophageal echocardiography in the perioperative period. Methods Thirty-one patients with mitral valve prolapse underwent mitral valve repair using chordae tendineae replacement concomitant with implantation of valveplasty ring. A 4-0 Goretex sutures was used for reconstruction of artificial chordae. Realtime three-dimensional transesophageal echocardiography was performed in all the patients during the preoperative, intraoperatire, and postoperative periods. The length of the chordae tendineae under the A1 section of the anterior leaflet and the P1 section of the posterior leaflet were measured and considered the normal length of chordae tendineae by real-time three-dimensional transesophageal echocardiography preoperatively. These pre-determined normal chordal lengths helped intraoperatively to approximate the length of the artificial chordae used and postoperatively to gauge the success of the procedures. The same values were used again postoperatively to gauge the success of intervention. Full flexible valveplasty rings were used in all the patients.Results There was no operative death. The mean cardiopulmonary bypass (CPB) and aortic cross clamp time were ( 142. 0 ±31.2 ) min and (98.0 ± 22.5 ) min, respectively. One patient' s intraoperative echocardiography upon termination of CPB showed persistent severe mitral regurgitation and was converted to mitral valve replacement. This patient was not included in the study group. The mean number of artificial chordae per patient was (2.0 ± 1.5 ) , range from 1 to 3. The mean preoperatively measured normal chordal length was ( 21.0 ± 2.5 ) mm, and the mean postoperative artificial chordal length was ( 20.0 ± 2.2 )mm. The difference was not significant. The follow-up interval was from 3 to 30 months and the follow-up rate was 98%. During the follow-up period, there was no late death. Trace mitral regurgitation (MR) was detected in 15 patients, mild and moderate MR were detected in 1 for each. No severe MR was detected. The freedom from reoperation was 100% during follow-up.There were no documented artificial chordae ruptures. Conclusion Conclusion Artificial chordal replacement with Gore-tex suture in mitral valve repair in this group of patients with mitral valve prolapse appears to have satisfactory early and mid-term results. Real-time three-dimensional transesophageal echocardiography plays a critical role in this technique. Real-time threedimensional transesophageal echocardiography can exactly predict the length of artificial chordae, which is helpful to improve the outcomes of mitral valve repair. However, longer term follow-up and larger series are required to validate our findings.

Objective To construct a chimeric infectious clone of the fatal virulent strain SDLY 107, containing the gene fragments encoding 2A and 3B proteins of the mild virulent strain SDLY 1, and to establish a reverse genetic system platform for further investigation on virulence of enterovirus 71 strains. Methods The overlap PCR analysis was performed to obtain the gene fragments encoding 2A and 3B pro-teins of the mild virulent strain SDLY 1.The obtained gene fragments were digested and then cloned into a plasmid pMD19-T containing the full-length gene of SDLY 107 strain by using gene replacement strategy. The recombinant RNA was transfected into Vero cells for the preparation of recombinant virus particles.Sev-eral assays including the PCR, indirect immunofluorescence ( IFA) , Western blot and sequencing were per-formed for virus identification.Virus titers were measured by 50%cell culture infective dose ( CCID50 ) and plaque assay.Results The infectious clones of SDLY 107-2A-1 and SDLY 107-3B-1 chimeric virus strains were constructed successfully.Typical cytopathic effect was observed in Vero cells after viral transfection. Identification of the rescued viruses by PCR, IFA, Western blot and sequencing further confirmed the suc-cessful construction of infectious virus strains.The virus titers of SDLY 107-2A-1 and SDLY 107-3B-1 strains detected by CCID50 and plaque assay were 1.25 ×105 PFU/ml and 0.7 ×105 PFU/ml, respectively. Conclusion The chimeric viruses SDLY 107-2A-1 and SDLY 107-3B-1 were rescued successfully, causing cytopathic effects similar to those by using the parental virus strain SDLY 107.This study might pave the way for further investigation on in vitro and in vivo virulence of enterovirus 71 strains.

Objective:To analyze the whole genome traceability and variation analysis of SARS-CoV-2 in local COVID-19 outbreaks in Binhai New Area, Tianjin.Methods:The whole-genome high-throughput sequencing was performed on throat swab samples collected from one local asymptomatic infected person and five confirmed cases of COVID-19 in Binhai New Area of Tianjin from November 7 to December 5, 2020. The sequencing data were assembled by De novo. MAFFT v7.0 multiple sequence alignment program and MEGA X software were used to compare the above data and construct phylogenetic tree (Neighbor-joining method).Results:The genetic similarity between the sequences of 6 SARS-CoV-2 strains and Wuhan reference sequence (Wuhan-Hu-1) was greater than 99.9%. Two of six strains were genetically identical, conform to the L-Lineage European Branch Ⅱ.1(America Branch)/B.1; The other four strains had the same genes and were in line with the characteristics of L-Lineage European Branch Ⅰ/B.1.1.These six strains belonged to different evolutionary branches and two different transmission chains. There were 18 nucleotide mutation sites in sequences of six SARS-CoV-2 strains, eight of which were synonymous mutation sites, nine of which were missense mutation sites, resulting in nine amino acid mutation sites, and important mutation sites of RDRP-P323L and S-D614G were found in all of the six samples.Conclusions:In this study, there were two COVID-19 outbreaks in Binhai New Area of Tianjin, and the sequences of six SARS-CoV-2 strains belonged to different evolutionary branches and two different transmission chains. It might come from porters′ contact with imported cold chain items contaminated with SARS-CoV-2 from different sources. All the sequences of six SARS-CoV-2 strains had P323L and D614G mutations, which indicated that the virus mutation and transmission ability were stronger. The surveillance of important employees of the cold chain in Tianjin and local and imported cases should be continuously strengthened.

Objective:To evaluate the efficacy and safety of sodium hyaluronate gel in preventing adhesion after prophylactic enterostomy.Methods:One hundred and twenty four patients from 6 hospitals were enrolled in this prospective multi-center randomized controlled trial. Patients were randomized into the study group ( n=59) or the control group ( n=65).All patients underwent prophylactic enterostomy. Patients of study group received odium hyaluronate gel for adhesion-prevention,while those in control group did not receive any adhesion-prevention treatment. The incidence of moderate to severe adhesion around the incision in the stoma area were evalutated during stoma reduction surgery. Results:The incidence of moderate to severe adhesion around the incision in the stoma area was 6.3% in the study group, the difference was statistically significant ( P<0.05) compared to that of the control group (32.6%). Conclusion:Sodium hyaluronate gel can safely and effectively reduce the incidence of moderate and severe adhesions after abdominal surgery.

Objective: To evaluate the immune responses and protection against human metapneumovirus (hMPV) conveyed by influenza virus vectors carrying multiple epitope antigens of hMPV. Methods: Two recombinant influenza viruses (rFLU/hMPV/B and rFLU/hMPV/CTL+ Th) carrying hMPV multi-epitope gene segments in NS gene were generated by reverse genetic techniques of eight-plasmid system. BALB/c mice were immunized intranasally with rFLU/hMPV/B and rFLU/hMPV/CTL+ Th twice at a two-week interval. Virus-specific antibody titers and splenocyte cytokines were detected two weeks after the boost immunization. Viral loads in lung tissues and turbinates were detected with digital PCR after the immunized mice were challenged with hMPV and influenza virus. Moreover, HE staining was used to observe lung injuries. Results: Specific antibodies against both the influenza virus and hMPV were induced in mice immunized intranasally with rFLU/hMPV/B, while the influenza virus-specific antibody response and hMPV-specific cytotoxic lymphocyte response (significant IFN-γ secretion) were detected in mice immunized with rFLU/hMPV/CTL+ Th. Additionally, balanced Th1/Th2 responses were elicited by rFLU/hMPV/B and rFLU/hMPV/CTL+ Th. Both rFLU/hMPV/B and rFLU/hMPV/CTL+ Th conveyed effective protection against subsequent influenza virus and hMPV challenges with significantly alleviated histopathological damages and reduced viral loads. Conclusions Both rFLU/hMPV/B and rFLU/hMPV/CTL+ Th can induce specific humoral immune response against hMPV and/or the influenza virus. Moreover, rFLU/hMPV/CTL+ Th can also elicit hMPV-specific CTL immune response. These two recombinant strains can also protect BALB/c mice from the challenges with hMPV and influenza virus, suggesting that they are promising vaccine candidates.