Clinical application of metagenomic next-generation sequencing in acquired immunodeficiency syndrome patients with pulmonary infections / 中华传染病杂志

Objective:To investigate the pathogen spectrum of acquired immunodeficiency syndrome (AIDS) patients with pulmonary opportunistic infections in the local area, and to evaluate the clinical application of metagenomic next-generation sequencing (mNGS) in these patients.Methods:From January to December 2021, AIDS patients with pulmonary infections admitted to Zhongnan Hospital of Wuhan University were enrolled. Their bronchoalveolar lavage fluid (BALF) was subjected to mNGS and coventional pathogen detection.Routine pathogen detection methods included smear, culture, polymerase chain reaction (PCR), and immunochromatographic colloidal gold. Fisher′s exact probability method was used for statistical analysis.Results:A total of 69 patients were included, and all of them were tested positive for mNGS. Among them, 53 cases (76.8%) were positive for fungi and viruses, 40 cases (58.0%) were positive for bacteria (excluding Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM)), six cases were positive for MTB, 11 cases were positive for NTM, and seven cases were positive for other pathogens. Mixed infections with two or more pathogens were found in 89.9%(62/69) of the patients. Among the conventional pathogen detections of BALF, 79.7%(55/69) of the patients were positive for pathogens, including 42 cases positive for Pneumocystis jirovecii PCR, 16 cases positive for BALF culture, nine cases positive for MTB PCR, and five cases positive for Cryptococcus antigen. The total detection rate of mNGS was 100.0%(69/69), which was higher than that of the conventional pathogen detection rate of 79.7%(55/69), and the difference was statistically significant (Fisher′s exact probability method, P<0.001). The specificity of mNGS detection was 88.4%. Combining clinical and two detection methods, the top five pathogens were Pneumocystis jirovecii (62.3%(43/69)), Candida (29.0%(20/69)), MTB (20.3%(14/69)), NTM and Talaromyces marneffei (15.9%(11/69), each). Fifty-three patients (76.8%) had co-infection with virus. Conclusions:The main cause of pulmonary infection in AIDS patients in this area is mixed infection, and Pneumocystis jirovecii is the most common pathogen. mNGS could significantly improve the pathogen detection rate in AIDS patients with pulmonary infections.

Feasibility of programmed death-1 monoclonal antibody treatment for patients with acquired immunodeficiency syndrome complicated with malignant tumor / 中华传染病杂志

Objective:To investigate the feasibility, efficacy and adverse reactions of programmed death-1(PD-1) inhibitors in patients with acquired immunodeficiency syndrome (AIDS) complicated with malignant tumor.Methods:From September 2020 to August 2021, patients with AIDS complicated with malignant tumor in Zhongnan Hospital of Wuhan University were enrolled. Data including basic information, laboratory test results, CD4 + T cell count, human immunodeficiency virus (HIV) viral load were collected. Patients were continuously administered intravenously PD-1 monoclonal antibody until disease progression or intolerant toxicity reaction occurred. Adverse reactions during treatment were recorded.And treatment outcomes were assessed once every 12 weeks after treatment. HIV viral load was measured after treatment once a week for four consecutive times, then once four weeks for two consecutive times, and then once every 12 weeks. Results:Ten patients were included in the study, including seven males and three females, three cases of Hodgkin′s lymphoma, two cases of cervical cancer and hepatocellular carcinoma respectively, one case of non-Hodgkin′s lymphoma, non-small cell lung cancer and anal cancer respectively. There were four patients with CD4 + T cell count of 100 to 200 cells/μL and two patients with CD4 + T cell count lower than 100 cells/μL. All patients had completed at least three cycles of treatment with PD-1 monoclonal antibody, HIV viral load remained lower than 20 copies /mL. Three cases achieved complete response and three cases achieved partial response. Adverse reactions were cutaneous capillary endothelial proliferation (CCEP) (seven cases), major bleeding (three cases), and hearing impairment (one case). Conclusions:PD-1 inhibitor has no adverse effect on the continuous suppression of HIV viral load and has an effect on tumor control, so it is a viable choice in AIDS patients complicated with tumor. However, due to its considerable adverse reactions, multidisciplinary cooperation is needed to reduce the risk of complications and deal with serious complications.

Relationship between masculinity and femininity and parental rearing pattern in lower grade primary school students / 中国学校卫生

Objective@#To analyze the correlation between the scores of masculinity and femininity and parental rearing pattern in lower grade primary school students in Xiamen, and to provide a policy suggestions and reference basis for establishing a correct concept of gender role among children and adolescents.@*Methods@#A cluster random sampling method was used to select 823 students from two primary schools in Xiamen. Masculinity and femininity scores were assessed by Children s Sex Role Inventory(CSRI), while attitudes and behaviors of parental rearing pattern were obtained through EMBU. Regression analyses were used to analyze the correlation between masculinity and femininity scores and parental rearing pattern.@*Results@#Masculinity scored 2.82 ( 2.41 ,3.24) and 2.82 (2.47,3.18), femininity scored 2.87 (2.40,3.20) and 3.13 (2.73,3.47) among boys and girls, with no significant gender difference ( P >0.05). Masculinity and femininity scores varied significantly by parental emotional warmth and understanding(father: Z/H =44.61, 37.24；mother: Z/H=41.68, 46.64, P <0.05). Among boys, increasing parental emotional warmth and understanding and paternal excessive interference were associated with higher masculinity and femininity scores. Increasing rejection and maternal deny were associated with lower masculinity scores. Among girls, increasing the understanding of emotional warmth of fathers was associated with masculinity and femininity scores, increasing excessive interference from mothers was associated with lower masculinity scores ( P <0.05).@*Conclusion@#There are gender differences in the relationship between parental rearing pattern and masculinity and femininity scores. In particular, increasing parents emotional warmth and understanding and reducing mothers punishment and harshness, rejection and denial, and other negative parenting styles could facilitate healthy development of masculinity and femininity among primary school students.

Clinical characteristics of 203 discharged patients with corona virus disease 2019 / 中华传染病杂志

Objective:To analyze the clinical data of 203 discharged patients with corona virus disease 2019(COVID-19), and to investigate the predictors for the severe cases.Methods:Confirmed COVID-19 cases hospitalized at Zhongnan Hospital of Wuhan University from January 1 to February 1, 2020 were consecutively enrolled, who were divided into severe group and non-severe group.The clinical data of enrolled patients were collected and the clinical manifestations, laboratory results, imaging, treatments and prognosis of patients in the two groups were analyzed. Mann-Whitney U rank sum test and chi-square test were used for statistical analysis. Results:A total of 203 discharged patients with COVID-19 were enrolled. The common clinical manifestations included fever (89.2%, 181/203), dry cough (60.1%, 122/203), chest distress (35.5%, 72/203), shortness of breath(29.1%, 59/203)and myalgia or arthralgia (26.6%, 54/203). The time from disease onset to hospital admission was 5.8 days (1.0 to 20.0 days). Among 203 enrolled patients, 107(52.7%) were divided into severe group and 96(47.3%) were non-severe group. The age in severe group was 60 years (23 to 91 years), which was significantly older than non-severe group (47 years (20 to 86 years)), the difference was statistically significant ( Z=-6.12, P<0.01). There were 63.6%(68/107) patients in severe group with at least one underlying disease, which was significantly more than non-severe group (20.8% (20/96)), the difference was statistically significant ( χ2=37.60, P<0.01). The proportions of patients with increased white blood cells, decreased lymphocytes and albumin, elevated alanine aminotransferase, aspartate aminotransferase, creatinine, lactic acid dehydrogenase, creatine kinase, fasting blood glucose, D-dimer, erythrocyte sedimentation rate, C-reactive protein, interleukin-6, and procalcitonin in severe group were all higher. On admission, 172 patients (84.7%) had bilateral patchy shadows or ground glass opacity in the lungs on chest imaging study, 20(9.9%) presented pleural effusion. Fifty-five cases (27.1%) showed progressions of lung lesions on computed tomography (CT) rescan at an average interval of five days. Among 203 patients, 123(60.6%) were given oxygen therapy upon admission, 107(52.7%) were given short-term glucocorticoid therapy, and 131(64.5%) received antiviral therapy; and 26(12.8%) died. The hospital stay was 11.0 days (1.0 to 45.0 days). Conclusions:Fever is the most common symptoms in COVID-19 patients.Elderly and patients with underlying diseases are risk factors for progression to severe cases. The elderly patients should be strengthened early monitoring, paid attention to the control of underlying diseases, and reduce the occurrence of critical diseases.

Association between nutritional status and puberty onset in boys / 中国学校卫生

Objective@#To explore the relationship between nutritional status and puberty onset in boys, and to provide a reference for promoting the development of physical and mental health of boys.@*Methods@#A total of 2 724 boys aged 7 to 12 years from grade 2 to 6 were recruited from Xiamen city by cluster sampling method in 2017. The nutritional status was assessed by physical examination, pubertal developmental status was evaluated by rating scales of Tanner and Prader orchidometer, and puberty timing was determined by the P25 age of puberty onset. The association between nutritional status and puberty onset was estimated by logistic regression model.@*Results@#Pubertal onset was found in 29.0% of the boys and the incidence of early pubertal timing was 2.9%. The prevalence of puberty onset in wasting, normal weight, overweight and obesity boys was 19.6%, 28.7%, 34.4% and 31.5%, respectively. The age of puberty onset was significantly earlier in obese boys (F=3.23, P<0.05). The results of Logistic regression analysis showed that with the increase of BMI, the possibility of puberty onset and risk of early pubertal timing increased. After adjusting for confounding factors, the odds of puberty onset in boys with wasting decreased by 64.0% (OR=0.36, 95%CI=0.22-0.60), the possibility of puberty onset and risk of early pubertal timing in boys with obesity increased by 78.3% (OR=1.78, 95%CI=1.14-2.79) and 192.9% (OR=2.93, 95%CI=1.46-5.86), respectively. These relationships were more pronounced in boys of households with lower economic level (P<0.05).@*Conclusion@#BMI was positively correlated with puberty onset in boys, the odds of puberty onset and risk of early pubertal timing were significantly increased in obese boys, especially in those with low household economic level.

Efficacy of platinum-based doublet chemotherapy versus single-agent chemotherapy as second-line treatment in elderly patients with advanced non-small cell lung cancer / 中华老年医学杂志

Objective To compare the efficacy and tolerance of platinum-based doublet chemotherapy versus single-agent chemotherapy as second-line treatment in elderly patients with advanced non-small cell lung cancer(NSCLC).Methods A total of 85 elderly patients with advanced NSCLC after first-line treatment were retrospectively analyzed and divided into the combination therapy group(n=40,taking platinum-based doublet chemotherapy)and the single-agent chemotherapy group(n=45,receiving single-agent second-line chemotherapy).Results There were no significant differences in the objective response rate (ORR)and the disease control rate (DCR)between the combination therapy group and the single-agent chemotherapy group(27.5 ％ or 11/40 vs.20.0 ％ or 9/45,60.0％ or 24/40 vs.73.3％ or 33/45,x2 =0.662 and 1.704,P=0.416 and 0.192).The median progression-free survival(PFS)was 3.8 months for the combination therapy group and 2.8 months for the single-agent chemotherapy group(P =0.045).The rate of grade Ⅲ/Ⅳ hematological toxicity was higher in the combination therapy group than in the single-agent chemotherapy group.Conclusions Platinum-based doublet chemotherapy has longer PFS than the single-agent chemotherapy as secondline treatment in elderly patients with advanced non-small cell lung cancer,and more attention should be paid to its high hematological toxicity.

Efficacy of first generation EGFR-TKIs and chemotherapy as first-line therapy in advanced lung adenocarcinoma patients with uncommon EGFR mutations / 中华肿瘤杂志

Objective@#To investigate the clinical effects of first generation epithelial growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) compared with platinum-based chemotherapy as first-line therapy in advanced lung adenocarcinoma patients with uncommon EGFR mutations.@*Methods@#Clinical data of 4 276 patients diagnosed as advanced lung adenocarcinoma (ⅢB/Ⅳ) underwent EGFR gene detection at the Affiliated Cancer Hospital of Zhengzhou University from January 2012 to February 2018 were collected and 99 cases with uncommon EGFR mutations were selected. The clinical pathological features, treatment outcomes, treatment options and prognosis after first-line treatment of the 99 cases were analysed and compared with other patients with common EGFR mutations.@*Results@#The objective response rates of patients with uncommon EGFR mutations receiving EGFR-TKIs or platinum-based chemotherapy were 33.0% and 27.1%, respectively. The disease control rates were 76.5% and 87.5%, respectively. The progression-free survival (PFS) of patients treated with EGFR-TKIs was 7.2 months, significantly superior than 4.9 months of patients receiving chemotherapy (P=0.009). The overall survival of patients treated with EGFR-TKIs was 14.3 months, significantly worse than 20.7 months of patients receiving chemotherapy (P=0.034). Multivariate analysis showed that distant metastases (P=0.001) and smoking history (P=0.013) were independent prognostic factors for OS of lung adenocarcinoma patients with EGFR uncommon mutations.@*Conclusions@#Compared with chemotherapy, the usage of first generation of EGFR-TKIs as first-line therapy can improve the short-term efficacy of advanced lung adenocarcinoma patients with EGFR uncommon mutations. However, platinum-based chemotherapy shows a longer overall survival.

Efficacy and Safety of Bevacizumab Combined with Chemotherapy as Second-line or Later-line Treatment in Advanced Nonsquamous Non-small Cell Lung Cancer / 中国肺癌杂志

BACKGROUND@#Bevacizumab combined with platinum-based chemotherapy has been recommended as the first-line agent in advanced nonsquamous non-small cell lung cancer (NSCLC) without driven gene, but this regimen is not common in the second-line or later-line treatment of non-squamous NSCLC. The aim of this study is to investigate the efficacy and safety of bevacizumab combined with chemotherapy as second-line or later-line treatment in advanced non-squamous NSCLC.@*METHODS@#We retrospectively reviewed the clinical data of advanced nonsquamous NSCLC patients who were treated with bevacizumab after first-line treatment failure and they were hospitalized in the Affiliated Cancer Hospital of Zhengzhou University from January 2014 to June 2017, and Kaplan-Meier method, Log-rank test and Cox model were used for analysis.@*RESULTS@#A total of 62 patients were included in the analysis. The total objective response rate (ORR) was 32.2%, and the disease control rate (DCR) was 96.8%. The median progression-free survival (PFS) was 6.4 months (95%CI: 6.05-6.83), and the median overall survival (OS) was 20.4 months (95%CI: 12.98-27.76). In the subgroup analysis, there was no significant difference in median PFS between patients with brain metastases and those without brain metastases (6.2 months vs 6.4 months, P=0.052). Cycles of bevacizumab (>6 or ≤6 cycles) was an independent influencing factor of PFS (P=0.004). The most common adverse events were leukopenia, fatigue, nausea, thrombocytopenia and hypertension.@*CONCLUSIONS@#In the second-line or later-line treatment, bevacizumab combined with chemotherapy is an effective and safe regimen for advanced non-squamous NSCLC.

Significance of The Maryland Aggregate Pathology Index in the evaluation of donation after citizens death (DCD) kidney by time-zero renal biopsy / 中华器官移植杂志

Objective To investigate the significance of The Maryland Aggregate Pathology Index in the evaluation of donation after citizens death (DCD) kidney by time-zero renal biopsy.Methods 124 kidney grafts were donated by 62 donors after cardiac death in First Hospital of Jilin University between Jan.2015 and Dec.2015.One kidney was deprecated after evaluation and 123 transplants were performed eventually.Time-zero renal biopsy was performed on 123 cases of DCD donor kidney,and rapid frozen pathological examination was performed.The pathological results of donor kidneys were graded by The Maryland Aggregate Pathology Index:low risk group (less than 7 points) (n =112 cases);the middle risk group (8-11 points) (n =11 cases),high the risk group (more than 12 points) (n =0).The incidence of delayed graft function (DGF),the incidence of perioperative acute rejection (AR),and the average creatinine level in the patients at different time points one year post-transplantation were observed.The median value of follow-up was 19 months,and the 1-year survival rate of patients and renal grafts was observed.Results All 123 cases of kidney transplantation from DCD were performed successfully.The incidence of DGF in low risk group and in middle risk group was 6.3％ (7/112) and 27.3％ (3/11),respectively (P =0.046).The incidence of perioperative acute rejection (AR) in low risk group and middle risk group was 9.8％ (11/112) and 27.3％ (3/11),respectively (P =0.112).The mean serum creatinine (Scr) levels at 7th day,1st month,3rd month,and 12th month after operation were 123.3 ± 79.7,104.4 ± 52.6,72.9 ± 32.0 and 107.6 ± 34.6 μmol/L in low risk group,and 321.0 ± 74.3,172.6 ± 59.9,142.9 ± 45.7 and 140.8 ± 63.6 μmol/L in middle risk group,respectively.The mean Scr levels in patients at different time points one year post-transplantation in low risk group were significantly lower than those in middle risk group (P＜0.000 1,=0.000 3,＜0.000 1,=0.012 respectively).The 1-year survival rate of patients and renal grafts was 98.2％ (10/112)/98.2 (110/112 in low risk group,and 81.8％ (9/11)/81.8％ (9/11) in middle risk group,respectively (P =0.040).Conclusion The Maryland aggregate pathology index obtained from time-zero renal biopsy of rapid frozen pathological examination can provide some guidance for the evaluation of the quality of DCD and the prognosis.Incidence of DGF was lower in low risk group than that in middle risk group,and the renal function of each time point was better within 1 year,and the 1-year survival rate of patients and renal grafts was higher.

Analysis of factors influencing delayed graft function following kidney transplants from donation after citizens death / 中华器官移植杂志

Objective To investigate the factors influencing delayed graft function (DGF)following kidney transplants from donation after citizens death (DCD) in single transplant center.Methods The clinical data of 504 kidney transplants from DCD in the First Hospital of Jilin University between August,2011 and May,2017 were collected and analyzed retrospectively.The functional recovery of kidney graft and the related factors were analyzed,respectively.Results The DGF occurred in 32 cases among 504 kidney transplant recipients during perioperative period,who received dialysis treatment (6.3％).The average recovery time of DGF was 21.0 ± 17.1 days.The average dialysis duration (41.3 ± 38.2 months) pre-transplant in DGF group was significantly longer than that in non-DGF group (28.9 ± 26.2 months) (P =0.024).The average age of donors (42.7 ±9.4 years) in DGF group was significantly older than that in non-DGF group (39.0 ± 15.9 years) (P=0.009).The ratio of donors who received CPR treatment in DGF group (21.9％) was significantly higher than that in non-DGF group (6.4％) (P =0.001).The average last serum creatinine level of donors in DGF group (149.3 ± 98.3 mol/L) was significantly higher than that in non-DGF group (92.8 ± 41.6 mol/L) (P＜0.001).The multivariate analysis revealed that CPR treatment for donors before organ donation,the last serum creatinine level of donors as well as BMI of kidney transplant recipients were all independent risk factor influencing DGF (P =0.001,P＜0.001 and P =0.008,respectively).Conclusion Our study focused on analysis of factors influencing DGF following kidney transplants from DCD in single transplant center.The results of this study are helpful to find and avoid the high risk factors that could influence DGF,reduce the incidence of DGF,improve the quality of patients' life as well as reduce the cost of medical treatment.

Generation and epitope mapping of a monoclonal antibody against nucleoprotein of Ebola virus / 生物工程学报

Ebola virus (EBOV) causes highly lethal hemorrhagic fever in humans and nonhuman primates and has a significant impact on public health. The nucleoprotein (NP) of EBOV (EBOV-NP) plays a central role in virus replication and has been used as a target molecule for disease diagnosis. In this study, we generated a monoclonal antibody (MAb) against EBOV-NP and mapped the epitope motif required for recognition by the MAb. The MAb generated via immunization of mice with prokaryotically expressed recombinant NP of the Zaire Ebola virus (ZEBOV-NP) was specific to ZEBOV-NP and able to recognize ZEBOV-NP expressed in prokaryotic and eukaryotic cells. The MAb cross-reacted with the NP of the Reston Ebola virus (REBOV), the Cote-d'Ivoire Ebola virus (CIEBOV) and the Bundibugyo Ebola virus (BEBOV) but not with the NP of the Sudan Ebola virus (SEBOV) or the Marburg virus (MARV). The minimal epitope sequence required for recognition by the MAb was the motif PPLESD, which is located between amino acid residues 583 and 588 at the C-terminus of ZEBOV-NP and well conserved among all 16 strains of ZEBOV, CIEBOV and BEBOV deposited in GenBank. The epitope motif is conserved in four out of five strains of REBOV.

Development of HBsAg-Binding Aptamers that bind HepG2.2.15 cells via HBV surface antigen / 中国病毒学·英文版

Hepatitis B virus surface antigen(HBsAg),a specific antigen on the membrane of Hepatitis B virus (HBV)-infected cells,provides a perfect target for therapeutic drugs.The development of reagents with high affinity and specificity to the HBsAg is of great significance to the early-stage diagnosis and treatment of HBV infection.Herein,we report the selection of RNA aptamers that can specifically bind to HBsAg protein and HBsAg-positive hepatocytes.One high affinity aptamer,HBs-A22,was isolated from an initial 115 mer library of ～1.1×1015 random-sequence RNA molecules using the SELEX procedure.The selected aptamer HBs-A22 bound specifically to hepatoma cell line HepG2.2.15 that expresses HBsAg but did not bind to HBsAg-devoid HepG2 cells.This is the first reported RNA aptamer which could bind to a HBV specific antigen.This newly isolated aptamer could be modified to deliver imaging,diagnostic,and therapeutic agents targeted at HBV-infected cells.

Characterization of M2 gene of H3N2 subtype swine influenza virus / 生物工程学报

M2 protein of influenza A virus is encoded by a spliced mRNA derived from RNA segment 7 and plays an important role in influenza virus replication. It is also a target molecule of anti-virus drugs. We extracted the viral genome RNAs from MDCK cells infected with swine influenza A virus (SIV) H3N2 subtype and amplified the SIV M2 gene by reverse transcriptase-polymerase chain reaction using the isloated viral genome RNAs as template. The amplified cDNA was cloned into a prokaryotic expression vector pET-28a(+) (designated pET-28a(+)-M2) and a eukaryotic expression vector p3xFLAG-CMV-7.1 (designated p3xFLAG-CMV-7.1-M2), respectively. The resulted constructs were confirmed by restriction enzyme digestion and DNA sequencing analysis. We then transformed the plasmid pET-28a(+)-M2 into Escherichia coli BL21 (DE3) strain and expressed it by adding 1 mmol/L of IPTG (isopropyl-beta-D-thiogalactopyranoside). The recombinant M2 protein was purified from the induced bacterial cells using Ni(2+) affinity chromatography. Wistar rats were immunized with the purified M2 protein for producing polyclonal antibodies specific for it. Western blotting analysis and immunofluorescence analysis showed that the produced antibodies were capable of reacting with M2 protein expressed in p3xFLAG-CMV-7.1-M2-transfected cells as well as that synthesized in SIV-infected cells. We also transfected plasmid p3xFLAG-CMV-7.1-M2 into Vero cells and analyzed its subcellular localization by immunofluorescence. The M2 protein expressed in the Vero cells was 20 kDa in size and dominantly localized in the cytoplasm, showing a similar distribution to that in SIV-infected cells. Western blotting analysis of SIV-infected cells suggested that M2 was a late phase protein, which was detectable 12 h post-infection, later than NS1, NP and M1 proteins. It would be a potential molecular indicator of late phases replication of virus. Our results would be useful for studying the biological function of M2 protein in SIV replication.

Establishment of a functional cell line expressing both subunits of H1a and H2c of human hepatocyte surface molecule ASGPR / 华中科技大学学报（医学）（英德文版）

To better understand the effect of a new split variant of human asialoglycoprotein receptor (ASGPR H1b) on ASGPR ligands' binding ability, we established a functional cell line which expresses ASGPR. The full lengths of ASGPRH1a and H2c fragments from human liver were amplified by reverse transcript PCR (RT-PCR) and inserted into eukaryotic expression vector pIRES2EGFP, pCDNA3.1 (Zeo+) respectively. The recombinants were co-transfected into HeLa cells. After selection by using Neocin and Zeocin, a stably transfected cell line was established, which was designated 4-1-6. The transcription and expression of ASGPRH1a and H2c in 4-1-6 were confirmed by RT-PCR, Western blotting and immunofluorescence. The endocytosis function of the artificial "ASGPR" on the surface of 4-1-6 was tested by FACS. It was found that the cell line 4-1-6 could bind ASGPR natural ligand molecular asialo-orosomucoid (ASOR). After the eukaryotic plasmid H1b/pCDNA3.1 (neo) was transfected into cell line 4-1-6, H1b did not down-regulate the ligand binding ability of ASGPR. The eukaryotic expression plasmid H1b/pcDNA3.1 (neo) and H2c/pcDNA3.1 (neo) were co-transfected transiently into Hela cell. Neither single H1b nor H1b and H2c could bind ASOR. In conclusion, a functional cell line of human asialoglycoprotein receptor (ASGPR) which expresses both H1a and H2c stably was established. The new split variant H1b has no effect on ASGPR binding to ASOR. ASGPRH1b alone can't bind to ASOR, it yet can't form functional complex with ASGPRH2c.

Cloning and characterization of M1 gene of H3N2 subtype swine influenza virus / 生物工程学报

Influenza A virus matrix protein (M1) is encoded by a spliced mRNA derived from RNA segment 7 and plays an important role in the virus life cycle. In the present study, we extracted the viral genome RNAs from allantoic fluid of 9-day-old embryonated chicken eggs infected with swine influenza A virus (SIV) H3N2 subtype and amplified the SIV M1 gene by reverse transcriptase-polymerase chain reaction using the isloated viral genome RNAs as template. The amplified cDNA was cloned into an expression vector pET-28a (+) (designated pET-28a-M1) and confirmed by DNA sequencing analysis. We then transformed the plasmid pET-28a-M1 into Escherichia coli BL21 strain for heterologous expression. The expression of M1 was induced by 1mM isopropyl-beta-D-thiogalactopyranoside. SDS-PAGE analysis of the induced bacterial cells revealed that the recombinant M1 protein was expressed in high yield level. Next, we purified the expressed recombinant M1 using Ni2+ affinity chromatography and immunized Wistar rat with the purified M1 protein for producing polyclonal antibodies specific for M1. Western blotting analysis showed that the produced antibodies were capable of reacting with M1 protein expressed in Escherichia coli as well as that synthesized in SIV-infected cells. We further cloned the amplified M1 cDNA into a eukaryotic expression plasmid p3xFLAG-CMV-7.1 to construct the recombinant plasmid p3xFLAG-CMV-M1 for expressing M1 in eukaryotic cells. Western blotting analysis revealed that the M1 protein was expressed in p3xFLAG-CMV-M1-transfected Vero cells and recognized by the produced anti-M1 antibodies. Using the produced anti-M1 antibodies, we analyzed the kinetics of M1 protein in the virus-infected cells during influenza virus infection and estimated the possibility of M1 as an indicator of influenza virus replication. The recombinant M1 protein, anti-M1 antibodies and recombinant expression plasmids would provide useful tools for studies of biological function of M1 protein and the basis of SIV replication.

Protective effect of short-term exercise on ischemic/reperfused myocardium in rats / 基础医学与临床

Objective To investigate the protective effect of short-term moderate exercise on ischemic/reperfused myocardium and its correlation to the activation of protein kinase C(PKC).Methods Fourty Wistar rats were randomly divided into 4 groups(n=10 respectively): control group(CON group),exercise group(EXE group),exercise + PKC inhibitor group(E+C group) and PKC inhibitor group(CHE group).The occurrence of arrhythmia,the recovery of cardiac function,and infarct size were observed by using the Langendorff-ischemia/reperfusion model in isolated rat heart in vitro.Results Recovery rate of LVDP(on the 30th and 60th minute of reperfusion) and RPP(on the 20th,30th and 60 minute of reperfusion) of EXE group were higher than those of CON and E+C groups(P