Objective:To construct recombinant influenza viruses expressing Gaussia luciferase (Gluc) with different influenza virus backbones and analyze their growth characteristics, genetic stability, ability to express Gluc and in vitro anti-influenza drug activity. Methods:The C-terminal of PR8NA was modified by inserting the porcine teschovirus-2A autocleavage peptide (P2A) and the Gluc-coding gene. Recombinant viruses, PR8NAGluc/PR8 and PR8NAGluc/WSN, were rescued using the eight-plasmid system of influenza virus reverse genetics, with seven plasmids derived from A/Puerto Rico/8/34(PR8) (H1N1) and A/WSN/1933 (WSN) H1N1. The genetic stability of the recombinant viruses was verified by RT-PCR. The fluorescence activity and the growth kinetics of the two recombinant viruses were compared. The correlation between the fluorescence activity of PR8NAGluc/WSN and median tissue culture infective dose (TCID 50), and the anti-drug activity of PR8NAGluc/WSN against oseltamivir, favipiravir, and Lianhua Qingwen in vitro were also analyzed. Results:The Gluc-expressing recombinant viruses constructed using PR8 and WSN backbones were successfully rescued by reverse genetics. Compared with the PR8 backbone, the WSN backbone significantly improved the fluorescence activity of Gluc. Moreover, the PR8NAGluc/WSN virus expressed stably in embryonated egg, and its replication kinetics was slightly lower than that of wild type. The fluorescence activity of PR8NAGluc/WSN virus had a good correlation with its TCID 50. The PR8NAGluc/WSN virus was sensitive to oseltamivir, favipiravir and Lianhua Qingwen. Conclusions:The recombinant virus with a WSN backbone exhibited higher fluorescence expression intensity as compared with the recombinant virus with a PR8 backbone. This study provided reference for high-throughput screening of anti-influenza drugs and the development of influenza virus vector vaccines.

Objective:To develop a recombinase-aided amplification (RAA)-clustered regularly interspaced short palindromic repeats(CRISPR)/Cas12a-based nucleic acid assay for monkeypox virus with high specificity and sensitivity.Methods:RAA primers and CRISPR RNA (crRNA) were designed based on the known conserved regions of the monkeypox virus gene and synthesized, and specific crRNAs were screened using fluorescence detection. The sensitivity and specificity of the detection system were evaluated.Results:An RAA-CRISPR/Cas12a-based nucleic acid assay for monkeypox virus was developed with a sensitivity of 2.5 copies/reaction and high specificity without cross-reactivity with ectromelia virus and vaccinia virus.Conclusions:An RAA-CRISPR/Cas12a-based nucleic acid assay for monkeypox virus was established, which would provide a powerful tool for efficient, rapid and specific detection of monkeypox virus.

Objective:To evaluate the immunogenicity of a novel influenza virus mRNA vaccine based on conserved antigens delivered by lipopolyplex (LPP) platform in a mouse model.Methods:Four copies of genes coding for extracellular domain of matrix 2 protein (M2e) and nucleoprotein (NP) of influenza A virus were synthetized after codon optimization. The fusion antigens were transcribed in vitro and delivered by LPP platform, named as LPP-4M2eNP. Expression of M2e and NP in eukaryotic cells was detected by immunofluorescence assay (IFA). BALB/c mice were inoculated intramuscularly twice with 10 μg or 30 μg LPP-4M2eNP vaccine at an interval of four weeks. Antibody response was detected by ELISA and cellular-mediated immunity (CMI) was detected by enzyme-linked immunospot assay (ELISPOT). Results:IFA showed that NP and M2e were expressed correctly in eukaryotic cells. Single dose immunization could induce significant antigen (NP, M2e)-specific CMI and antigen (NP, M2e)-specific antibody response was induced in mice with Th1 type bias after boost immunization. Moreover, NP-specific CMI was increased significantly after the second immunization, while no significant change in M2e-specific CMI was observed.Conclusions:Stronger CMI was triggered in mice by single dose of LPP-4M2eNP vaccine. Furthermore, robust humoral and cellular immune responses were induced after boost immunization. This study suggested that LPP-4M2eNP vaccine, which based on conserved antigen of influenza A and delivered by LPP platform, had great potential for development and application.

Objective:To improve the consistency of test results through reducing inter-laboratory variation in SARS-CoV-2 antibody detection with WHO SARS-CoV-2 antibody candidate international standard (IS, sample G) and antibody reference panel (samples E, F, H, I, J).Methods:Ten WHO samples (A-J) including the candidate IS and reference panel were evaluated using different methods, such as microneutralization tests based on live SARS-CoV-2, pseudovirus neutralization assay and commercial ELISA kits. The test results were compared using statistical analysis.Results:Using IS (sample G) as a reference, the relative concentrations of other samples could be determined with less variation. ELISA and pseudovirus neutralization assay had consistent results with those obtained with the microneutralization test based on SARS-COV-2 strain HB02. Weakly positive samples could be detected only by a certain kit.Conclusions:The availability of an IS for antibodies would facilitate the standardization of SARS-CoV-2 antibody detection methods. The reference panel fitted all the assays based on the SARS-CoV-2 prototype Wuhan strain. Pseudovirus neutralization assay and ELISA could be used as alternatives to live SARS-CoV-2-based neutralization test to some extent.

Objective:To examine the correlation factors of contralateral central lymph node metastasis in unilateral papillary thyroid carcinoma with lateral cervical lymph node metastasis.Methods:The clinical data of patients with unilateral papillary thyroid carcinoma who underwent total thyroidectomy and bilateral central lymph node dissection and ipsilateral cervical lymph node dissection from June 2016 to June 2018 at Department of Thyroid Surgery, West China Hospital, Sichuan University were analyzed retrospectively. A total of 317 patients, including 87 males and 230 females, aged (41.4±12.1) years (range: 16 to 75 years), were enrolled in this study. The risk factors of contralateral central lymph node metastasis were analyzed by χ 2 test and Spearman correlation analysis. Results:There were 116, 69, 269, and 181 cases of pretracheal lymph node, prelaryngeal lymph node, ipsilateral central lymph node and contralateral central lymph node metastasis, respectively, and 16 cases of skipping metastasis. Univariate analysis showed that contralateral central lymph node metastasis was associated with gender, maximum tumor diameter, capsule invasion, pretracheal lymph node metastasis, prelaryngeal lymph node metastasis, and ipsilateral central lymph node metastasis (all P<0.05). Spearman correlation analysis showed that male ( r s=0.162, P=0.004), maximum tumor diameter>10 mm ( r s=0.184, P=0.001), capsule invasion ( r s=0.135, P=0.016), pretracheal lymph node metastasis ( r s=0.394, P<0.01), prelaryngeal lymph node metastasis ( r s=0.272, P<0.01) and ipsilateral central lymph node metastasis ( r s=0.203, P<0.01) were independent correlation factors for contralateral central lymph node metastasis. Conclusion:For patients with unilateral papillary thyroid carcinoma with ipsilateral cervical lymph node metastasis, bilateral central lymph node dissection should be considered if male, tumor diameter>10 mm, capsule invasion, pretracheal lymph node metastasis, prelaryngeal lymph node metastasis, or ipsilateral central lymph node metastasis.

Objective:To examine the correlation factors of contralateral central lymph node metastasis in unilateral papillary thyroid carcinoma with lateral cervical lymph node metastasis.Methods:The clinical data of patients with unilateral papillary thyroid carcinoma who underwent total thyroidectomy and bilateral central lymph node dissection and ipsilateral cervical lymph node dissection from June 2016 to June 2018 at Department of Thyroid Surgery, West China Hospital, Sichuan University were analyzed retrospectively. A total of 317 patients, including 87 males and 230 females, aged (41.4±12.1) years (range: 16 to 75 years), were enrolled in this study. The risk factors of contralateral central lymph node metastasis were analyzed by χ 2 test and Spearman correlation analysis. Results:There were 116, 69, 269, and 181 cases of pretracheal lymph node, prelaryngeal lymph node, ipsilateral central lymph node and contralateral central lymph node metastasis, respectively, and 16 cases of skipping metastasis. Univariate analysis showed that contralateral central lymph node metastasis was associated with gender, maximum tumor diameter, capsule invasion, pretracheal lymph node metastasis, prelaryngeal lymph node metastasis, and ipsilateral central lymph node metastasis (all P<0.05). Spearman correlation analysis showed that male ( r s=0.162, P=0.004), maximum tumor diameter>10 mm ( r s=0.184, P=0.001), capsule invasion ( r s=0.135, P=0.016), pretracheal lymph node metastasis ( r s=0.394, P<0.01), prelaryngeal lymph node metastasis ( r s=0.272, P<0.01) and ipsilateral central lymph node metastasis ( r s=0.203, P<0.01) were independent correlation factors for contralateral central lymph node metastasis. Conclusion:For patients with unilateral papillary thyroid carcinoma with ipsilateral cervical lymph node metastasis, bilateral central lymph node dissection should be considered if male, tumor diameter>10 mm, capsule invasion, pretracheal lymph node metastasis, prelaryngeal lymph node metastasis, or ipsilateral central lymph node metastasis.

Objective To observe the effects of phlorizin on aorta of diabetes db/db mice and to explore its mechanism.Methods Sixteen diabetes db/db mice were randomized into two groups:untreated diabetic group (DM group) and diabetic group treated by phlorizin(DMT group) with eight db/m mice as normal control group(CC group).Phlorizin (20 mg/kg) was given in normal saline solution intragastrically to the mice of the DMT group for 10 weeks.Mice of the other two groups were given the same amount of saline solution intragastrically for 10 weeks.Animals were weighed weekly.At 10th weekend,all mice were fasted overnight and then sacrificed.Fasting blood was collected,and aortas were dissected.The blood samples were analyzed for fasting blood glucose (FBG),triglycerides (TG),total cholesterol (TC),Serum advanced glycation end products(AGEs)and malondialdehyde (MDA).Aortic tissue were examined microscopically.Results At 10th weekend,the weight (57.53±3.40)g and serum concentration of FBG(31.21±2.16) mmol/L,TG (0.39±0.12) mmol/L,TC(3.15±0.30)mmol/L,AGEs (0.28±0.04) AU/mg and MDA (15.18± 1.60)mol/L in DM group were increased than those in CC group (P＜0.01),and the weight(54.24± 1.28)g,FBG(29.17±1.41) mmol/L,TG(0.26±0.06) mmol/L,TC(2.71±0.26) mmol/L,AGEs (0.24±0.03) AU/mg and MDA(13.46±1.28)mol/L were lowered significantly in DMT group than those in DM group (P＜0.05 or P＜0.01).The severity of aorta damage in DMT group was less than that in DM group.Conclusions Phlorizin can protect the db/db mice from diabetic macrovascular complications,which may be attributed to its decreasing of blood glucose,TG,TC,and AGEs levels,and its antioxidant potential.

Objective To compare the diagnostic efficacies of narrow-band imaging (NBI) in distinguishing neoplastic from non-neoplastic colorectal lesions with routine endoscopy and with magnifying endoscopy. Methods Patients with colorectal lesions detected by NBI from September 2008 to February 2010 were enrolled in the study. These lesions were classified by pit pattern and capillary pattern, which was then assessed by reference to histopathology. Results A total of 100 patients with colorectal lesions were enrolled, and the lesions were observed by NBI with ordinary endoscopy (n =64) and NBI with magnifying endoscopy (n =36), respectively, and 7 cases (5 in NBI with ordinary endoscopy and 2 in NBI with magnifying endoscopy) which did not meet the diagnostic criteria were excluded. The overall diagnostic accuracy of NBI endoscopy in distinguishing neoplastic from non-neoplastic colorectal lesions was 91.4% ( 85/93 ), in which NBI with ordinary endoscopy and magnifying endoscopy was 89. 8% (53/59) and 94. 1% (32/34),respectively, with both significantly higher than that of conventional colonoscopy reported in the literature (79. 1% ) (P ＜ 0. 05 ). However, no significant difference was detected between 2 methods ( P ＞ 0. 05 ).Conclusion Similar with NBI magnifying endoscopy, NBI endoscopy without high magnification may also be useful to distinguish neoplastic from non-neoplastic colorectal lesions.

potential use in clinical therapy and prognosis estimation of the tumor.

Objective To investigate whether tumor necrosis factor (TNF)a, TNFb and TNFc microsatellite polymorphisms correlate with chronic atrophic gastritis and gastric adenocarcinoma in Chinese Han population. Methods TNFa, TNFb and TNFc microsatellite alleles in 164 healthy subjects, 53 patients with chronic atrophic gastritis and 56 patients with gastric adenocarcinoma were typed using PCR technique combined with High Voltage denaturing PAGE and silver staining. At the same time, the PCR products were cloned and sequenced. Results The frequency of TNFa10 allele was significantly higher in patients with chronic atrophic gastritis than in healthy individuals ( 19.81% vs. 11.89% , P = 0.04 ). However it was not related to age, gender, degree of atrophy or intestinal metaplasia in patients with chronic atrophic gastritis. The frequency of TNFa6b5c1 haplotype homozygote was significantly lower in patients with gastric adenocarcinoma than in healthy individuals ( 1.79% vs. 15.85% , P = 0.006 ). The sequence result revealed that the copy number of dinucleiotide repeating within the same TNFa allele was not consistent with that in the reports from Western countries. Conclusions It should be more accurate and clear to define TNFa alleles. TNFa10 allele is associated with the susceptibility to chronic atrophic gastritis. TNFa6b5c1 haplotype homozygote is negatively associated with gastric adenocarcinoma and thus may play a resistant role in the shifting process from chronic atrophic gastritis to gastric adenocarcinoma.