Objective:To investigate the impact of inserting an exogenous gene, NanoLuc (Nluc), at different sites in the influenza virus genome on viral properties and analyze the expression stability of the exogenous gene both in vitro and in vivo. Methods:Using molecular cloning techniques and reverse genetics, eight recombinant influenza viruses were constructed by inserting the exogenous Nluc gene into the gene segments encoding hemagglutinin (HA), neuraminidase (NA), non-structural protein (NS), and polymerase basic protein 1 (PB1). Viral replication capacity was evaluated by hemagglutination and plaque assays. Nluc expression in infected cells was monitored by fluorescence imaging. The potential impact of the exogenous gene insertion on viral infectivity was examined in a mouse infection model. Independent samples t-test were used for statistical analysis. Results:The recombinant viruses with insertions in the HA, NA, and NS gene segments generated fluorescent signals in the first generation of rescued viruses and demonstrated replication capabilities in plaque and hemagglutination assays. The recombinant viruses based on the NA and NS genes were capable of stably expressing Nluc across different generations, and exhibited correct fluorescent distribution patterns in mouse infection experiments. Meanwhile, the NS gene-based recombinant virus demonstrated superior stability in the mouse model.Conclusions:This study demonstrates that the NS gene segment of influenza virus can serve as an effective insertion site for exogenous genes without impairing the viral replication or infectivity, and the recombinant virus constructed based on it exhibits high integration stability and substantial application potential.

Objective:To investigate the impact of inserting an exogenous gene, NanoLuc (Nluc), at different sites in the influenza virus genome on viral properties and analyze the expression stability of the exogenous gene both in vitro and in vivo. Methods:Using molecular cloning techniques and reverse genetics, eight recombinant influenza viruses were constructed by inserting the exogenous Nluc gene into the gene segments encoding hemagglutinin (HA), neuraminidase (NA), non-structural protein (NS), and polymerase basic protein 1 (PB1). Viral replication capacity was evaluated by hemagglutination and plaque assays. Nluc expression in infected cells was monitored by fluorescence imaging. The potential impact of the exogenous gene insertion on viral infectivity was examined in a mouse infection model. Independent samples t-test were used for statistical analysis. Results:The recombinant viruses with insertions in the HA, NA, and NS gene segments generated fluorescent signals in the first generation of rescued viruses and demonstrated replication capabilities in plaque and hemagglutination assays. The recombinant viruses based on the NA and NS genes were capable of stably expressing Nluc across different generations, and exhibited correct fluorescent distribution patterns in mouse infection experiments. Meanwhile, the NS gene-based recombinant virus demonstrated superior stability in the mouse model.Conclusions:This study demonstrates that the NS gene segment of influenza virus can serve as an effective insertion site for exogenous genes without impairing the viral replication or infectivity, and the recombinant virus constructed based on it exhibits high integration stability and substantial application potential.

Objective To systematically summarize the published literature on the genetic variants associated with nonalcoholic fatty liver disease(NAFLD). Methods Literature from Web of Science,PubMed,and Embase between January 1980 and September 2022 was systematically searched.Meta-analyses of the genetic variants were conducted using at least five data sources.The epidemiologic credibility of the significant associations was graded using the Venice criteria. Results Based on literature screening,399 eligible studies were included,comprising 381 candidate gene association,16 genome-wide association,and 2 whole-exome sequencing studies.We identified 465 genetic variants in 173 genes in candidate gene association studies,and 25 genetic variants in 17 genes were included in the meta-analysis.The meta-analysis identified 11 variants in 10 genes that were significantly associated with NAFLD,with cumulative epidemiological evidence of an association graded as strong for two variants in two genes(HFE,TNF),moderate for four variants in three genes(TM6SF2,GCKR,and ADIPOQ),and weak for five variants in five genes(MBOAT7,PEMT,PNPLA3,LEPR,and MTHFR). Conclusion This study identified six variants in five genes that had moderate to strong evidence of an association with NAFLD,which may help understand the genetic architecture of NAFLD risk.

Objective To screen a Madin-Darby canine kidney (MDCK) cell line for H5N1 influ-enza virus isolation and to evaluate its safety in vaccine production. Methods MDCK cells were cloned by the method of limiting dilution. Hemagglutination test was used to screen MDCK cells that were suitable for H5N1 influenza virus production. Tests for analyzing the characteristics, extraneous agents, endogenous agents and tumorigenicity of MDCK cells were performed according to Chinese Pharmacopeia Volume Ⅲ. Results A total of 108 MDCK cell lines were obtained and three of them were selected after hemagglutina-tion test. G1 cells were chosen following further screening with tumorigenicity test and receptor abundance analysis. The average number of chromosomes of the MDCK-G1 cells was 78±4. No bacteria, fungi or myco-plasma contamination was detected. In experimental group, each nude mouse was injected with 1×107/ml viable cells to observe their tumorigenicity. Twelve weeks after cell injection, no node was found at injection sites or in gross anatomy. There was no significant difference between the experimental and negative control groups. The result of the tumorigenicity test was negative. No node formation was found after injecting nude mice with cell lysate or cellular DNA collected from equivalent amount of cells. It was indicated that the MDCK-G1 cells were of low-oncogenic potential. Conclusions The MDCK-G1 cell line could be used as a substrate to produce H5N1 influenza virus vaccine.

Objective To evaluate the efficacy of interventional therapy via apical approach in complex mitral valve paravalvular leakage (PVL).Methods From January 2014 to December 2016,interventional therapy by apical approach was used in 7 patients with mitral valve PVL after mechanical valve replacement.There were 5 male and 2 female with mean age of 42-64 (51.3 ± 7.1) years.Six patients had a history of previous infective endocarditis.There were 3 cases of NYHA heart function =Ⅲ,and 4 cases =Ⅳ.The period of time between interventional therapy and previous operation was 6.5-8 (3.6 ± 3.1) years,with mitral regurgitation volume:9.5-23.1 (13.3 ± 4.7)ml.Interventional therapy of small incision method via the left sixth intercostal was carried out in the catheterization laboratory or the hybrid operation room with the patient under general anesthesia.Follow-up evaluation included peri-operational mortality,complications,improvement of cardiac function,hemolysis and postoperative residual mitral regurgitation.Results The success rate of total operation was 100％.The average operation time was 90-300 (145.7 ± 71.8) min,and the DSA radiation time was 6-25 (12.1 ± 6.5)min,with average hospitalization time of 5-12 (10.2 ± 3.5)d.The main post-operative complications included 1 case of hemoptysis,1 case of hematuria and acute renal failure,and 2 cases of blood transfusion,with blood transfusion volume of 1 200 ml and 3 290 ml,respectively.During the follow-up,there was no death.Mitral regurgitation volume decreased to 0 -1.0(0.43 ± 0.45) ml (P ＜ 0.05).All patients had improved heart function in different degree and no serious complications.Conclusions Interventional therapy via apical approach in complex mitral valve PVL has the advantages of being a simpler and safer approach,less trauma,shorter time of hospitalization,faster post-operative recovery and lower treatment cost.Its disadvantages are high difficult operation,strict indications and high technical requirements for the surgeon.By improving operation skills,choosing individualized occluders and treatment of early hemolytic complications,the success rate and long-term effect of interventional therapy can get further improved.

Objective To compare the safety and efficacy between transcatheter intervention and surgical closure of para-valvular leakage(PVL)after valve replacement.Methods From May 2010 to May 2014,17 patients of paravalvular leakage af-ter valve replacement underwent interventional therapy and 11 patients underwent surgical closure.The perioperative charac-ters, mortality rate, complications and prognosis were compared between the patients underwent surgical procedure and tran-scatheter intervention.The average follow-up time was 13 months(1-36 months).Results The success rate of transcatheter intervention was 100%, while there were 2 in-hospital deaths in the surgical group(18%).Compared with surgical group, transcatheter intervention group has the benefit of shorter operation time[(84 ±36)min vs.(358 ±88)min](P<0.01),shor-ter hospitalization time[(11.9 ±12.1)d vs.(38.1 ±42.2)d](P<0.05), less transfusion[(1 029 ±455)ml vs.(1 438 ± 908)ml](P<0.05).There was no mechanical ventilation and ICU stay in the transcatheter intervention group.After tran-scatheter intervention, there was 7 residual shunt(47%)with an average shunt volume of(1.6 ±1.7)ml,which is remarkably reduced compared with pre-operation.In the surgical group, there was 1 case of residual shunt(9%).During the follow-up, there was no deaths in both groups, with improved heart function and no serious complications.Conclusion In the treatment of paravalvular leakage after valve replacement,transcatheter interventiontechnique has the advantages of simpler and safe ap-proach, less trauma, shorter time of hospitalization,faster post-operative recovery and lower treatment cost.This method is one of the new technology in the field of minimally invasive cardiac surgery in recent years.By improving operation skills,choosing individualized occluders and treatment of early hemolytic complications, the success rate and long-term effect of interventional therapy can get further improved.

OBJECTIVETo assess the association of three polymorphisms (14-bpINS/DEL, +3035C/T and +3142C/G) in the 3' untranslated region (3'UTR) of HLA-G gene and systemic lupus erythematosus (SLE) in Yunnan.

METHODSA case-control study has been carried out on 206 SLE patients and 212 healthy controls. Genotypes of 14-bpINS/DEL (rs1704), +3035C/T (rs17179108) and +3142C/G (rs1063320) loci of 3'UTR of the HLA-G gene were determined with DNA sequencing.

RESULTSAllelic and genotypic frequencies of 14-bpINS/DEL and +3142C/G did not differ significantly between the two groups (P > 0.05). The frequencies of +3035T allele was significantly higher in the SLE group compared with the control group (P < 0.01, OR=1.604, 95% CI: 1.186-2.169). With a dominant inheritance model, the odd ratio of dominant genotype (CT+TT) was 2.004 (95% CI: 1.345-2.987, P=0.0006) in the SLE group.

CONCLUSIONThe 14-bpINS/DEL and +3142C/G polymorphisms in the 3'UTR of the HLA-G gene are not associated with susceptibility to SLE in Yunnan, whilst the T allele of +3035C/T may be a risk factor for SLE.

3' Untranslated Regions ; genetics ; Case-Control Studies ; China ; Female ; Genetic Predisposition to Disease ; HLA-G Antigens ; genetics ; Humans ; Lupus Erythematosus, Systemic ; genetics ; Male ; Polymorphism, Genetic

Between May 2009 and February 2012,60 patients scheduled for laparoscopic appendectomy were randomly assigned to receive either transumbilical single-port laparoscopic appendectomy (TUSPLA) or three-port laparoscopic appendectomy (TPLA).The operative duration was significantly longer in TUSPLA than TPLA cases.No statistically significant differences existed in hospital stay,visual analog scale (VAS) score,analgesia requirement or complication rate.The abdominal scar was inconspicuous for TUSPLA.It suggested that TUSPLA with conventional instrumentation is technically feasible and safe with scarless healing.

OBJECTIVETo explore the relationship between genetic polymorphisms of 3 single nucleotide polymorphisms (SNPs) in the elastin microfibril interfacer 1 (EMILIN1) gene and essential hypertension.

METHODSA case-control study was conducted in which 201 hypertensive patients and 202 healthy controls in Mongolian population were enrolled, and the genotypes of rs3754734, rs2011616 and rs2304682 loci were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and direct sequencing techniques.

RESULTSThere were significant differences in the frequencies of alleles and genotypes for the rs2304682 between the hypertensives and normotensives in the population (P<0.05). The frequency of the G-G haplotype established by rs3754734 and rs2304682 was significantly higher in the hypertensive patients (P<0.05). The frequencies of alleles and genotypes for the rs2304682 also had significant differences between the group with high diastolic blood pressure and normal diasto lic blood pressure (P<0.05).There were no significant differences in the frequencies of alleles and genotypes for the 3 SNPs between the group with high systolic blood pressure and normal systolic blood pressure (P>0.05).

CONCLUSIONThe rs2304682 locus in the EMILIN1 gene, as well as the haplotypes G-G constructed using rs3754734 and rs2304682, may associate with the susceptibility of essential hypertension in the Mongolian population. Also, rs2304682 may associate with the level of the diastolic blood pressure.

Blood Pressure ; genetics ; Case-Control Studies ; Genetic Predisposition to Disease ; Humans ; Hypertension ; genetics ; Membrane Glycoproteins ; genetics ; Middle Aged ; Mongolia ; Polymorphism, Single Nucleotide

BACKGROUND: How to make growth factor plays a role persistently and efficiently is a key in constructing bone tissue engineered bone. Currently, varied microspheres or scaffolds were used as release carriers, however, the delayed release effects needs elevating.OBJECTIVE: To prepare chitosan microosPheres/nano-hydroxyapatite/poly (lactic-co-glycolic acid) (CMs/nHA/PLGA) scaffolds, and to measure its characteristics of delayed release of bovine serum albumin (BSA).METHODS: CMs were prepared by an emulsifying cross linking method with BSA as a model protein. Using ice particulates as porogen, composite CMs/nHA/PLGA scaffolds were prepared by freeze-drying. The characteristic and morphology of the composite were observed by scanning electron microscope, later particle size analyzer, mercury porosimeter and universal testing machine, and the release behavior of BSA was investigated in vitro.RESULTS AND CONCLUSION: The CMs were spherical shape with a regular surface, with diameters of 20-40 μm. The encapsulation efficiency of the CMs was 86.5%, and the loading capacity was 0.8%. With the increase of initial BSA dosage, the loading capacity increased to 2.6%, while the encapsulation efficiency decreased to 74.1%. The CMs can be uniformly distributed in PLGA scaffolds to form CMs/nHA/PLGA scaffolds, which had 100-400 μm pore diameter and over 80% porosity, with 1.1-2.3 pMPa compressive strength, and 26.5% cumulative degradation at 10 weeks. The cumulative release of BSA from nHA/PLGA scaffolds was above 85% at 36 hours, which from CMs was 33.6% at 10 days, and that from CMs/nHA/PLGA scaffolds was 81.5% at 40 days. The results demonstrated that CMs/nHA/PLGA scaffolds have an excellent releasing efficiency for protein drugs with suitable compressive strength and degradation, which would be used as delivery system and tissue engineering scaffolds.