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.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Cercopithecus aethiops ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Influenza A Virus, H3N2 Subtype ; genetics ; RNA ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Swine ; Transfection ; Vero Cells ; Viral Matrix Proteins ; biosynthesis ; genetics ; Virus Replication ; genetics

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.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Chick Embryo ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Influenza A Virus, H3N2 Subtype ; genetics ; physiology ; Rats ; Rats, Wistar ; Recombinant Proteins ; genetics ; immunology ; metabolism ; Swine ; Viral Matrix Proteins ; genetics ; immunology ; metabolism ; Virus Replication ; genetics

Objective To study the effect of comprehensive treatment on elderly diabetes patients with early diabetic foot lesion .Methods Infrared thermography was used to check the temperature of double lower-limb skin in elderly patients with diabetes , then the temperature difference of both sides was compared .A total of 106 patients received comprehensive treatment of drug treatment intervention , physical intervention , health guidance, whose graphic showed the temperature difference .Then, observed the values of temperature difference , positive area, discomfort and blood glucose of patients before and after the intervention .Results Significant differences were showed in terms of the values of temperature difference [(1.91 ±1.21) vs (0.21 ±0.12)℃;t=8.817,P<0.01] positive area change situation of lower limbs [(9.90 ±5.52) vs (1.90 ±0.52)cm2;t=11.574,P<0.01], HbA1C [(7.90 ±1.32) vs (6.88 ±0.52)%;t=7.402,P<0.01]before and 3 months after intervention among 106 elderly diabetes patients with early diabetic foot lesion .Conclusions It was found that infrared thermography had a positive effect on finding abnormal imaging changes of the foot skin temperature in elderly patients with diabetes timely .And comprehensive treatment of drug treatment intervention , physical intervention , health guidance can effectively prevent diabetic foot complications happening .

Objective:To investigate urinary microalbumin to creatinine ratio (ACR) and α1-microglobulin to creatinine ratio (MCR) of people aged 40 years old and above in Shanxi province, and analyze the influencing factors of abnormal ACR and MCR, and to provide evidence for the prevention and control of chronic kidney diseases.Methods:It was a cross-sectional study. The data came from a screening study of chronic kidney diseases conducted by Shanxi Provincial People's Hospital from April to November 2019, involving aged 40 years old and above from 10 counties (Ningwu county, Yu county, Yangqu county, Lin county, Shouyang county, Zezhou county, Huozhou city, Hejin city, Linyi county and Ruicheng county) in Shanxi province. The related data were collected through questionnaire surveys, physical examinations, and blood and urine sample collection. Urinary α1-microglobulin, creatinine, and microalbuminuria were measured. Urinary ACR and MCR were calculated using urinary creatinine correction. ACR abnormality was defined as ≥30 mg/g, and MCR abnormality was defined as >23 mg/g. Covariate analysis was used to control confounding factors, and adjusted urinary ACR and MCR of 10 counties were calculated. Spearman correlation analysis and chi-square test were performed to analyze the factors associated with abnormal urinary ACR and MCR. Logistic regression analysis model was used to identify the influencing factors of abnormal urinary ACR and MCR.Results:A total of 12 285 residents were enrolled in the study, including 5 206 males (42.4%) and 7 079 females (57.6%). The median age was 58.0 (51.0, 66.0) years old. The median urinary ACR was 7.5 (4.5, 15.7) mg/g, and the median urinary MCR was 10.2 (6.4, 16.2) mg/g. A total of 1 572 individuals (12.80%) had urinary ACR abnormality and 1 450 individuals (11.80%) had urinary MCR abnormality. Yangqu county, Yuxian county, and Ningwu county had higher urinary ACR with (35.58±3.04) mg/g, (34.08±4.50) mg/g and (32.09±3.19) mg/g, respectively. The urinary MCR was generally similar among the 10 counties and Yangqu county had higher urinary MCR with (13.86±0.41) mg/g. In addition to Yu county, female individuals had higher urinary ACR compared to males in other counties, whereas female individuals had lower urinary MCR compared to males in 10 counties. Multivariate logistic regression analysis results showed that elevated triglyceride, fasting blood glucose, glycated hemoglobin, systolic blood pressure, diastolic blood pressure, age, body mass index and gender were independent influencing factors of abnormal urinary ACR and MCR (all P<0.05). Elevated blood homocysteine and low educational level were independent influencing factors of urinary MCR abnormality (both P<0.05). Conclusions:There are differences of gender and region in urinary ACR and MCR among individuals aged 40 years old and above in the 10 counties of Shanxi province. Triglyceride, fasting blood glucose, glycated hemoglobin, systolic blood pressure, diastolic blood pressure, age, gender, and body mass index are independent related factors of abnormal urinary ACR and MCR. Blood homocysteine and education level are independent related factors of abnormal urinary MCR.

Objective:To examine the early and mid-term results of coronary endarterectomy (CE) combined with coronary artery bypass grafting (CABG) in the treatment of diffuse coronary artery stenosis.Methods:The clinical data and follow-up results of 248 patients who underwent CE+CABG surgery from January 2010 to January 2019 at Department of Cardiovascular Surgery, Nanjing Hospital Affiliated to Nanjing Medical University was analyzed retrospectively. There were 201 males and 47 females, aged (65.6±8.5) years (range: 43 to 79 years). The on-pump group included 156 patients and the off-pump group included 92 patients. CABG was performed after CE. CE was performed on 248 patients who represented 269 target coronary lesions, of which 108 were located on the left anterior descending artery and sub-branches, 140 were located on the right coronary artery and sub-branches, and 21 were located on the left circumflex artery and obtuse marginal artery. A total of 872 bypass grafts were performed, including 248 left internal thoracic arteries, 48 radial arteries, and 576 great saphenous veins, with (3.5±0.8) grafts (range:2 to 6 grafts) per patient.The grafts had satisfactory bridge blood flow after CE, with the graft flow rates of (26±8) ml/min (range: 13 to 59 ml/min) and the pulsatility index value of 3.1±0.8 (range: 2.0 to 6.7). The t test and χ 2 test was used to compare the surgical results and graft patency rate between patients in on-pump and off-pump group, respectively. Results:The number of graft vessels of on-pump group and off-pump group was 3.6±0.9 and 3.2±0.7, respectively( t=1.637, P=0.085). There were 3 deaths during the perioperative period, with a mortality rate of 1.2%. Two people died of renal failure, and one case was due to postoperative refractory low cardiac output. Perioperative myocardial infarction occurred in 9 cases. The follow-up time was (41.8±21.4) months (range:1 to 68 months). The all graft patency rate was 78.4%(812/232) in 1 year and 69.8%(162/232) in 3 years postoperatively. The left coronary graft patency rate was significantly higher than the right coronary graft patency rate(1-year: 87.4% vs.73.1%, χ2=6.533, P=0.011, 3-year: 78.2% vs. 64.8%, χ2=4.588, P=0.032). There was no significant difference in graft patency rates between the on-pump group and off-pump group (1-year: 80.0% vs. 76.9%, χ2=0.277, P=0.599, 3-year:71.5% vs. 67.9%, χ2=0.300, P=0.584). Conclusions:CE+CABG is a safe and feasible technique for patients with diffuse coronary artery disease to get more satisfied complete revascularization, with good early and medium-term results and graft patency rates. The outcomes of on-pump or off-pump CE+CABG are similar.

Objective:To examine the early and mid-term results of coronary endarterectomy (CE) combined with coronary artery bypass grafting (CABG) in the treatment of diffuse coronary artery stenosis.Methods:The clinical data and follow-up results of 248 patients who underwent CE+CABG surgery from January 2010 to January 2019 at Department of Cardiovascular Surgery, Nanjing Hospital Affiliated to Nanjing Medical University was analyzed retrospectively. There were 201 males and 47 females, aged (65.6±8.5) years (range: 43 to 79 years). The on-pump group included 156 patients and the off-pump group included 92 patients. CABG was performed after CE. CE was performed on 248 patients who represented 269 target coronary lesions, of which 108 were located on the left anterior descending artery and sub-branches, 140 were located on the right coronary artery and sub-branches, and 21 were located on the left circumflex artery and obtuse marginal artery. A total of 872 bypass grafts were performed, including 248 left internal thoracic arteries, 48 radial arteries, and 576 great saphenous veins, with (3.5±0.8) grafts (range:2 to 6 grafts) per patient.The grafts had satisfactory bridge blood flow after CE, with the graft flow rates of (26±8) ml/min (range: 13 to 59 ml/min) and the pulsatility index value of 3.1±0.8 (range: 2.0 to 6.7). The t test and χ 2 test was used to compare the surgical results and graft patency rate between patients in on-pump and off-pump group, respectively. Results:The number of graft vessels of on-pump group and off-pump group was 3.6±0.9 and 3.2±0.7, respectively( t=1.637, P=0.085). There were 3 deaths during the perioperative period, with a mortality rate of 1.2%. Two people died of renal failure, and one case was due to postoperative refractory low cardiac output. Perioperative myocardial infarction occurred in 9 cases. The follow-up time was (41.8±21.4) months (range:1 to 68 months). The all graft patency rate was 78.4%(812/232) in 1 year and 69.8%(162/232) in 3 years postoperatively. The left coronary graft patency rate was significantly higher than the right coronary graft patency rate(1-year: 87.4% vs.73.1%, χ2=6.533, P=0.011, 3-year: 78.2% vs. 64.8%, χ2=4.588, P=0.032). There was no significant difference in graft patency rates between the on-pump group and off-pump group (1-year: 80.0% vs. 76.9%, χ2=0.277, P=0.599, 3-year:71.5% vs. 67.9%, χ2=0.300, P=0.584). Conclusions:CE+CABG is a safe and feasible technique for patients with diffuse coronary artery disease to get more satisfied complete revascularization, with good early and medium-term results and graft patency rates. The outcomes of on-pump or off-pump CE+CABG are similar.

Objective:To explore the challenges in the implementation of drug centralized volume-based procurement policies in hospitals and propose corresponding optimization suggestions.Methods:From August to December 2023, a purposive sampling was conducted to select 11 pharmaceutical experts from tertiary hospitals in China for Delphi method. The survey content included " policy recommendations for promoting the acceleration and expansion of national drug centralized procurement and retaining surplus medical insurance funds for centralized procurement" .Results:Survey participants gave feedback on a set of existing problems found in the implementation of drug centralized procurement policies and proposed corresponding optimization methods. Kendall′s W coefficient of the specialist consultation was 0.332( P<0.05), demonstrating good consistency and concentration of the expert opinions. Among the problems, the score of drug supply guarantee was the highest(mean value of importance was 4.45). At the same time, the recommendation of strengthening monitoring and early warning, coordination and dispatch, and effectively ensuring the supply of centralized drug procurement had the highest score and concentration(mean value of importance was 4.91, coefficient of variation was 0.06). Conclusions:Through Delphi method, this study revealed issues and optimization methods in the implementation of drug centralized procurement policies in hospitals. The findings could provide valuable insights for improvements in the pharmaceutical sector and future policy adjustments.