This study investigated the specific immune response of BALB/c mice that was induced by a circular RNA (circRNA) vaccine expressing the herpes simplex virus type II (HSV-2) glycoprotein D (gD). The aim was to evaluate the immunological potential of this vaccine and lay a foundation for developing an mRNA vaccine against HSV-2. PCR and homologous recombination were employed to integrate the gD gene obtained from the pT7AMP-gD ectodomain plasmid into pUC57 to generate the recombinant plasmid pUC57-circ-gD, which was then sequenced and characterized. In vitro transcription and cyclization were performed on the template DNA to generate pUC57-circ-gD mRNA. To validate the formation of circular RNA, we cleaved the pUC57-circ-gD mRNA with RNase R and employed RT-PCR to validate the cyclization. The pUC57-circ-gD mRNA was then transfected into 293T cells. After 72 h, the cell supernatant was collected, and Western blotting was employed to measure the protein level of gD. Subsequently, the mRNA was encapsulated in lipid nanoparticles (LNPs) by microfluidic encapsulation. BALB/c mice were administrated with the encapsulated mRNA, and blood was collected from the fundus venous plexus after 21 and 35 days, and from the enucleated eyeballs after 49 days. Enzyme-linked immunosorbent assay was employed to measure the titers of antibodies, including virus-neutralizing antibodies. After 49 days, spleens were harvested and assessed for secretion of interferon-gamma (IFN-γ) by solid-phase enzyme-linked immunospot. The results showed successful construction and sequencing of the recombinant plasmid. RNase R digestion confirmed the presence of circular RNAs. Western blotting of the 293T cells transfected with the mRNA showed clear specific bands. The quality of the vaccine was tested by size exclusion chromatography-high performance liquid chromatography, which showed that the purity of the vaccine was about 90%. The mRNA-LNP showcased the particle size of 82.76 nm and an encapsulation rate of approximately 98%. Following three-dose vaccination, all immunized mice exhibited steady weight gain with 100% survival rate throughout the 28-day observation period, indicating no significant acute toxicity associated with the vaccine formulation. The immunized mice showed dose-dependent increases in serum IgG antibody titer and IFN-γ secretion by splenocytes and they were resistant to virus attacks. These findings indicate good immunogenicity and persistence of the pUC57-circ-gD mRNA vaccine, providing a reference for further studies on circRNA vaccines.
Animals ; Mice, Inbred BALB C ; RNA, Circular ; Mice ; Humans ; Herpesvirus 2, Human/genetics* ; Viral Envelope Proteins/genetics* ; Antibodies, Viral/blood* ; HEK293 Cells ; Female ; Nanoparticles ; Plasmids

Objective:To discuss the effect of modified ultrafiltration combined with sequential infusion of blood products, such as platelets and cryoprecipitation, on perioperative coagulation function in neonates undergoing cardiovascular surgery under extracorporeal circulation.Methods:A retrospective analysis was performed on 83 neonates who underwent cardiovascular surgery from January 2017 to December 2019. They were divided into the control group (conventional ultrafiltration, 51 cases) and the treatment group (modified ultrafiltration + sequential infusion of blood products, 32 cases).Results:The age of treatment group was significantly younger than that of the control group ( P<0.05). The extracorporeal circulation time of the treatment group was significantly longer than that of the control group. Compared with the preoperative data, post-APTT, post-PT and post-INR were increased significantly in the control group, platelet count and FIB were decreased significantly ( P<0.01), while there was no significant difference in the treatment group. Compared with the treatment groups, post-APTT, post-PT and post-INR were significantly increased in the control group, and postoperative platelet count and FIB were significantly decreased ( P<0.05). The drainage of control group was significantly higher than that of the treatment group at 12 h and 24 h after surgery ( P<0.01). Conclusion:The application of modified ultrafiltration combined with sequential infusion of blood products can significantly improve perioperative coagulation and reduce mediastinal bleeding in neonatal after cardiovascular surgery.

Objective:To review and analyze the clinical experience of children with aortic valve stenosis and/or insufficiency treated with autologous pulmonary valve for aortic valve replacement procedure(Ross operation) with ePTFE artificial valve for right ventricular outflow tract reconstruction.Methods:From 2015 to 2020, 8 cases of aortic stenosis and/or aortic insufficiency treated by Ross operation in our center were collected, with an age of 0.5-13.2 years old. 4 cases of aortic stenosis were diagnosed preoperatively, 3 cases of aortic stenosis with aortic insufficiency, and 1 case of infective endocarditis involving the aortic valve. The operation was carried out in three steps: Harvest autologous pulmonary valve; the diseased aortic valve was resected and autologous pulmonary valve was transplanted to the aorta by aortic root transplantation; the right ventricular outflow tract was reconstructed by a handmade ePTFE artificial flap blood vessel.Results:In 6 cases, the right ventricular outflow tract was reconstructed by hand-sewn ePTFE trileaflets, and artificial univalve in 2 cases, no death occurred during operation; all patients were cured and discharged. The patients were followed up for 1 to 36 months, with mean of(12.63±12.19) months. There was no long-term death or valvular complications. During follow-up echocardiography indicated 1 case of moderate aortic regurgitation, 1 case of mild-moderate regurgitation, and moderate regurgitation was found in 2 patients with artificial single pulmonary valve. For the remaining patients, they were mild aortic regurgitation, and a trivial or mild pulmonary artery regurgitation with hand-sewn three-leaflets ePTFE artificial vessel; All patients were followed up at the last time with a peak pressure of(6.63±3.46) mmHg(1 mmHg=0.133 kPa) across the aortic valve. The left ventricular outflow tract and aortic annulus shrank slightly after surgery(the diameter of one patient with Ross-Konno operation increased), but the annulus diameter increased with age. There was no need for further intervention.Conclusion:The Ross operation is safe for the treatment of aortic valve disease, it has good hemodynamic effect, and the autologous pulmonary artery has growth potential, especially suitable for children and young patients. Hand-sewn ePTFE with trileaflet vessels for reconstruction of right ventricular outflow tract performs well in anti-regurgitation function in the short term or may be used as a replacement material for the homograft/heterograft vessel, but longer follow-up and more cases are needed.

Objective:To investigate the effects of short-term pulmonary rehabilitation on the exercise ability and life quality of men with acute exacerbations of chronic obstructive pulmonary disease (COPD).Methods:Thirty-eight male COPD patients with acute exacerbation were selected and randomly divided into a rehabilitation group ( n=20) and a control group ( n=18). Both groups were given routine health education and medication, while the rehabilitation group was additionally provided with a 7-day, three-stage rehabilitation intervention, including the respiratory training, resistance training using a resistance band and gradual low-intensity walking training, all under remote electrocardiographic monitoring. Before and after the intervention, both groups′ exercise ability was evaluated using the 30-second chair sitting test (30-STS) and a 30-second forearm load flexion test (30-ACT). Each subject′s quality of life and ability in the activities of daily living were evaluated by two qualified rehabilitation therapists. The subjects′ level of daily physical activity was assessed 2 months after discharge. Results:After the intervention the average 30-STS and 30-ACT results of the rehabilitation group had improved significantly. The 30-ACT results of the control group were also significantly improved. After the intervention the average 30-STS and 30-ACT results of the rehabilitation group were significantly better than the control group′s averages. The average CAT and MBI scores of both groups were significantly better after the intervention, with the rehabilitation group′s averages significantly better than those of the control group. Two months after discharge, the number of patients with at least moderate daily physical activity increased from 6 to 19 in the rehabilitation group and from 4 to in the control group, a significant difference between the two groups.Conclusion:The three-stage pulmonary rehabilitation program is helpful for improving the exercise ability and quality of life of COPD sufferers, and their ability in the activities of daily living. Their level of daily physical activity is higher 2 months after discharge.

Objective To review the clinical experience with the Norwood stage Ⅰprocedure.Methods Between June 2016 to October 2018, totally 5 neonates underwent Norwood stage Ⅰ procedure.There were 3 boys and 2 girls, weighing(2. 98 ±0.60)kg with median of 2.95 kg.Age at surgeries ranged from 1 to 8 days.All 5 cases underwent the Norwood stage Ⅰprocedure under deep hypothermic circulatory arrest, including 3 cases of modified Blalock-Taussig shunts (MBTS) and 2 ca-ses of RV-PA shunt.Results The third case was successfully closed the chest on postoperative day 2 and extubated, but died from DIC due to severe infection on the postoperative day 6.The fifth case died from pericardiol tamponade at 10 hours after the operation.The first,second and fourth cases were followed up 1 month after discharge with NYHA Ⅰ, but the first and second cases dropped out of follow-up due to some personal resaons.The fourth case underwent the bidirectional Glenn procedure 9 monthes after the stage Ⅰ procedure and recovered smoothly.Conclusion The application of these modified methods and te-chiques based on the classical Norwood procedure help to accumulate experience in sugical treatment of HLHS in China .

Objective: To observe the autonomic nerve functional changing and the impact of comprehensive exercise in patients with cardiovascular neurosis (CN). Methods: Our research included in 2 groups: CN group, n=48 and Control group, n=30 normal subjects. Resting heart rate (RHR) and heart rate at the first recovery minute (HRR1) were measured by cardiopulmonary exercise testing (CPET) for comparison between 2 groups. In CN group, 30 patients were chosen and randomly assigned into 2 subgroups: Medication subgroup, patients received β-receptor blocker, n=14 and Comprehensive exercise subgroup, patients received the same medication plus aerobic and Thera-band resistance training, n=16; both subgroups were intervened for 3 months. Symptom checklist 90 (SCL-90) was used to evaluate relevant parameters before and after intervention, the changes of RHR and HRR1 were also compared between 2 subgroups. Results: ① Compared with Control group, CN group had the higher RHR and lower HRR1, all P<0.01. ② Compared with pre-intervention, both subgroups had improved RHR, HRR1 and SCL-90 scores at post-intervention, all P<0.05; compared with Medication subgroup, Comprehensive exercise subgroup showed improved SCL-90 scores and HRR1, all P<0.05. Conclusion: Comprehensive exercise including aerobic and Thera-band resistance training could effectively improve the clinical symptoms in CN patients, which might be related to changing the autonomic nerve function in relevant patients.

Objective: To observe the effects of aerobic exercise combined with resistance training on the cardiorespiratory fitness and exercise capacity of patients with stable coronary artery disease (CAD) . Methods: From June 2014 to December 2015, 73 patients with stable CAD in our department were recruited and randomly assigned to two groups: the control group (n=38) and the exercise group (n=35) . Patients in both groups received conventional medical treatment for CAD and related cardiac health education. While for patients in exercise group, a twelve-week aerobic exercise combined with resistance training program were applied on top of conventional treatment and health education. Cardiorespiratory fitness and exercise capacity were evaluated by cardiopulmonary exercise testing. Results: (1) The exercise capacity was significantly increased in the exercise group after 12 weeks training as compared to baseline level: peak oxygen uptake per kilogram ( (26.25±5.14) ml·kg^-1·min^-1 vs. (20.88±4.59) ml·kg^-1·min^-1) , anaerobic threshold ( (15.24±2.75) ml·kg^-1·min^-1 vs. (13.52±2.92) ml·kg^-1·min^-1], peak oxygen pulse ( (11.91±2.89) ml/beat vs. (9.77±2.49) ml/beat) , peak Watts ( (113.2±34.0) W vs. (103.7±27.9) W) , peak metabolic equivalent ( (7.57±1.46) METs vs. (6.00±1.32) METs) (all P<0.05 vs. baseline) . (2) The degree of improvement of peak oxygen uptake per kilogram ( (26.25±5.14) ml·kg^-1·min^-1 vs. (22.32±4.00) ml·kg^-1·min^-1) , anaerobic threshold ( (15.24±2.75) ml·kg^-1·min^-1 vs. (13.76±2.51) ml·kg^-1·min^-1) , peak oxygen pulse ( (11.91±2.89) ml/beat vs. (9.99±2.15) ml/beat) and peak metabolic equivalent ( (7.57±1.46) METs vs. (6.47±1.17) METs) were significantly higher in exercise group than in control group (all P<0.05) . Conclusion Aerobic training at an aerobic threshold level combined with Thera-band resistance training is safe for patients with stable coronary artery disease. This combined exercise program can significantly improve the cardiorespiratory fitness and exercise capacity of patients with stable coronary artery disease.

Objective To observe the influence of exercises on the mRNA expression of peroxisome proliferator-activated receptor (PPAR-α) and its target genes of acyl-CoA oxidase (ACO),Enoyl-CoA-hydratase and 3-hydroxyacyl-CoA-dehydrogenase (EHHADH) in the skeletal muscles in insulin-resistance mice to develop a way to improve the lipid metabolism.Methods Twenty male ApoE knockout mice were randomly divided into two groups,the high-fat diet group (group HFD) and the exercise training group (group Ex).The HFD group were fed with highfat diet,while the Ex group were fed in the same way,with additionally swimming training.And ten healthy male C57BL/6j mice were chosen as the control group(group ND).After 12 weeks of intervention,the serum lipid,blood glucose and insulin levels were determined,and homeostasis model assessment insulin resistance index (Homa-IRI) was calculated.The bilateral gastrocnemiuses were cut to be observed under a transmission electron microscope,and the mRNA expression of PPAR-α,ACO and EHHADH in skeletal muscle were measured using reverse transcription polymerase chain reaction(RT-PCR).Results The transmission electron microscope showed that the sarcolemma edema,mitochondrial swelling,as well as focal myocytolysis and edema within myofibrils were observed in the HFD group.The total cholesterol,low-density lipoprotein cholesterol,free fatty acid,fasting glucose,insulin and HomaIRI of the HFD group were significantly higher than the control group (P ＜ 0.05),while the mRNA expression of PPAR-α,ACO and EHHADH was significantly deceased than the latter(P ＜ 0.05).After swimming,the abovementioned pathological changes disappeared.The serum lipid of the Ex group were significantly lower (P ＜ 0.05),while HDL was significantly higher (P ＜ 0.05).And fasting insulin,glucose and HOMA-IR of the Ex group were significantly lower (P ＜ 0.05),while the mRNA expression of the above in the Ex group were significantly increased (P ＜0.05).Conclusion Swimming training could improve insulin resistance and metabolic disorder of lipid of ApoE knockout mice.The possible mechanisms may be through up-regulating the expression of PPAR-α,which in turn stimulates the expression of ACO and EHHADH mRNA to strengthen fatty acid β-oxidation.

Objective To investigate the serum leptin, leptin receptor of tissue in rats with insulin resistance (IR), and the effect of exercise on them. Methods 9 male Sprague-Dawley rats were fed with normal diet as control group. 34 rats were randomly assigned into 4 groups: model group (n=9), fed with high-fat/high-sucrose diet without any other intervention; metformin group (n=8), fed with high-fat/high-sucrose diet and metformin 300 mg/kg·d; exercise group (n=9), fed with high-fat/high-sucrose diet and trained with swimming; integrated group (n=8), fed with high-fat/high-sucrose diet and metformin 300 mg/kg·d, and trained with swimming. After 6 weeks of intervention,the serum leptin was measured with the enzyme-linked immunoabsorbent assay; the expression of leptin receptor protein in liver, skeletal muscle and adipose were measured with immunohistochemistry, and the expression of leptin receptor mRNA in the same tissues was measured with reverse transcriptase-polymerase chain reaction. Results Compared with the model group, the levels of fasting blood glucose and fasting insulin decreased in the exercise, metformin and integrated group (P<0.05), while the IR index increased (P<0.05). Compared with the control group, the serum leptin increased in the model group (P<0.01), while the expression of leptin receptor protein and mRNA in liver, skeletal muscle and adipose decreased (P<0.01). Compared with the model group, the serum leptin decreased in the exercise, metformin and integrated groups (P<0.05), while the expression of leptin receptor protein and mRNA increased (P<0.05). Compared with the metformin group, the expression of leptin receptor protein and mRNA increased in the exercise and integrated groups (P<0.05). Conclusion High level of serum leptin and low expression of leptin receptor can be found in rats with insulin resistance. Exercise can improve the insulin resistance, which may associate with the decrease of the serum leptin and increases of the leptin receptor.