[Establishment and preliminary application of quantitative real-time PCR assay for the detection of SARS-CoV-2 subgenomic nucleocapsid RNA].

Objective: To establish a rapid and specific quantitative real-time PCR (qPCR) method for the detection of SARS-CoV-2 subgenomic nucleocapsid RNA (SgN) in patients with COVID-19 or environmental samples. Methods: The qPCR assay was established by designing specific primers and TaqMan probe based on the SARS-CoV-2 genomic sequence in Global Initiative of Sharing All Influenza Data (GISAID) database. The reaction conditions were optimized by using different annealing temperature, different primers and probe concentrations and the standard curve was established. Further, the specificity, sensitivity and repeatability were also assessed. The established SgN and genomic RNA (gRNA) qPCR assays were both applied to detect 21 environmental samples and 351 clinical samples containing 48 recovered patients. In the specimens with both positive gRNA and positive SgN, 25 specimens were inoculated on cells. Results: The primers and probes of SgN had good specificity for SARS-CoV-2. The minimum detection limit of the preliminarily established qPCR detection method for SgN was 1.5×102 copies/ml, with a coefficient of variation less than 1%. The positive rate of gRNA in 372 samples was 97.04% (361/372). The positive rates of SgN in positive environmental samples and positive clinical samples were 36.84% (7/19) and 49.42% (169/342), respectively. The positive rate and copy number of SgN in Wild strain were lower than those of SARS-CoV-2 Delta strain. Among the 25 SgN positive samples, 12 samples within 5 days of sampling time were all isolated with virus; 13 samples sampled for more than 12 days had no cytopathic effect. Conclusion: A qPCR method for the detection of SARS-CoV-2 SgN has been successfully established. The sensitivity, specificity and repeatability of this method are good.

[The current status of endovascular aortic arch repair].

Endovascular aortic arch repair provides treatment opportunity for patients with aortic arch dissection and aneurysm who are intolerant to open surgery. The aortic arch branches provide the blood flow for brain, the revascularization of these branches is part and parcel of the endovascular aortic arch repair. The anatomical configuration, high-speed blood flow and long access from femoral artery increase the difficulty of endovascular aortic arch repair. Debranch technique, combined with chimney, scallop, and fenestration, have partially simplified the endovascular aortic arch repair. The dedicated endografts for aortic arch is becoming a research focus. In the design of aortic arch endografts, fenestrated and branched stent-grafts are the two main strategies. A variety of innovative concepts have been applied in the design of aortic arch endografts, including modular and integrated design, inner branch and outer branch design, single branch and multi-branch design, etc. Today, these procedures of complex endovascular aortic arch repair still need to be limited to experienced centers. Endovascular aortic arch repair showed favorable short-term outcomes through the development of strict surgical plans, as well as effective teamwork. Long-term efficacy and safety in larger participants need further investigation.

[Underlying mechanism and preventive measures of distal stent graft-induced new entry after thoracic endovascular aortic repair for Stanford type B aortic dissection].

Distal stent graft-induced new entry (dSINE) is the complication with high incidence following thoracic endovascular aortic repair (TEVAR) for Stanford type B aortic dissection. This review the underlying mechanism and preventive measures regarding dSINE. General mechanism include: the fragile state of dissected aortic wall and intimal flap is pathophysiologic foundation of dSINE; the continued resistive force to the deformation of stent-graft is mechanical cause of intimal injury; the intimal flap movement within a cardiac cycle result in local damage accumulation in distal site of stent-graft. Aortic remolding play an important role in prophylaxis of dSINE.

Biological function of microRNA-30c/SOX9 in pediatric osteosarcoma cell growth and metastasis.

OBJECTIVE: Osteosarcoma is one of the commonest malignant bone tumors, which frequently occurs in children all over the world. To find out methods to improve the therapeutic effect of osteosarcoma, it is necessary to detect the functioning mechanism of miR-30c to regulate the proliferation and metastasis of osteosarcoma cell. PATIENTS AND METHODS: In order to reveal the expression level of miR-30c, quantitative Real-time PCR (qRT-PCR) method was chosen. To evaluate cell viability and proliferation rates, colony formation and cell counting kit-8 (CCK8) assay were introduced. Based on cell migration and invasion assay, metastasis capacity of breast cancer cells was studied. Protein levels were measured by Western blotting assay and cell cycle distribution was identified by flow cytometry. Bioinformatics analysis and Luciferase assay were used to predict and verify the target gene. RESULTS: Compared with pericarcinomatous tissues (n=38), miR-30c in osteosarcoma tissues was significantly suppressed. Overexpressed miR-30c could weaken osteosarcoma cell's abilities of viability, proliferation, migration and invasion. Moreover, it could also encourage osteosarcoma cell apoptosis and block cell cycle at G0/G1 phase. According to bioinformatics analysis and Luciferase reporter assay, SOX9 was recognized as the target gene of miR-30c. Restoration of SOX9 could make miR-30c regain the ability of suppression on tumorigenesis of osteosarcoma cells. CONCLUSIONS: MiR-30c could play an important role in tumor suppression for pediatric osteosarcoma development and metastasis by targeting SOX9 in vitro. Thus, a creative and potential target was provided for diagnosis and treatment of osteosarcoma.

[Effect of ultrasound guided patient-controlled paravertebral block on pulmonary function in patients with multiple fractured ribs].

Objective: To investigated the effects of patient controlled intravenous analgesia and ultrasound guided patient-controlled paravertebral block on pulmonary function in patients with multiple fractured ribs. Methods: From May 2015 to March 2016, 60 patients with multiple rib fractures in Ningbo NO.6 Hospital with American Society of Anesthesiologists Ⅱ-Ⅲ underwent internal fixation, including 30 males and 30 females aged from 16 to 70 years(mean age (54.6±15.2) years). All patients were randomly divided into 2 groups (n=30 each): patient controlled intravenous analgesia(PCIA) group and patient controlled thoracic paravertebral block(PCPB) group.PCIA solution contained sufentanil 2 µg/kg in 100 ml of normal saline.The PCIA pump was set up to deliver a 2 ml bolus dose with 15 minutes lockout interval and background infusion at 2 ml/hour.In PCPB group, the patients received paravertebral injection of 0.2% ropivocaine 15 ml on the corresponding area of fractured side under ultrasound guidance at the end of operation, and then received PCPB.PCPB solution contained 0.75% ropivacaine 67 ml in 250 ml of normal saline, and the pump was set up to deliver a 5 ml bolus dose, with 15 minutes lock out interval and background infusion at 5 ml/hour. Both analgesia lasted to 72 hours after operation.Before the operation, at 30 minutes, 60 minutes, 1 day, 2 days, 3 days after analgesia, arterial blood samples were collected to test the levels of pH, PaO2, PaCO2, PaO2/FiO2 and PA-aDO2. The pulmonary function was also examined before and 3 days after the operation through FEV1 and FEV1%. Results: Compared with PCIA group at 30 minutes, 60 minutes, 1 day, 2 days, 3 days after analgesia, the level of PaO2 ((85.1±9.7)mmHg vs.(72.4±12.3)mmHg, (90.3±12.4)mmHg vs.(73.5±7.8)mmHg, (94.2±8.2)mmHg vs.(86.1±5.6)mmHg, (98.5±7.0)mmHg vs. (88.8±7.5)mmHg, (99.6±9.8)mmHg vs. (91.3±8.6)mmHg, P<0.05)) and PaO2/FiO2 were significantly increased ((405.1±46.0)mmHg vs. (340.1±58.9)mmHg, (430.5±59.1)mmHg vs. (344.0±65.4)mmHg, (448.3±39.1)mmHg vs. (410.1±26.7)mmHg, (460.1±33.5)mmHg vs. (423.2±36.5)mmHg, (465.1±28.2)mmHg vs. (435.1±40.8)mmHg, P<0.05)), the level of PA-aDO2 was decreased ((22.9±4.6)mmHg vs. (36.6±5.1)mmHg, (17.7±4.7)mmHg vs. (34.5±2.9)mmHg, (13.8±4.1)mmHg vs. (21.9±3.2)mmHg, (13.5±4.6)mmHg vs. (19.2±3.8)mmHg, (12.4±2.0)mmHg vs. (17.7±2.4)mmHg, P<0.05)), and FEV1, FEV1% were significantly increased at 3 days after operation in PCPB group ((2.9±0.4)mmHg vs.(2.2±0.5)mmHg, (78.1±4.7)mmHg vs.(64.8±4.3)mmHg; P<0.01)). Conclusion: Ultrasound guided patient-controlled paravertebral block improves the arterial oxygenation function and accelerates the recovery of pulmonary function in patients with multiple-fractured ribs after internal fixation operation.

[Reconstruct abdominal aortic branch arteries totally by endovascular repair in complex aortic aneurysms: a single-centre experience].

OBJECTIVE: To report experiences and results of complex aortic aneurysms involving the visceral arteries treating by total endovascular repair. METHODS: Seventy cases of thoracoabdominal aortic lesions treating by total endovascular repair in Department of Vascular Surgery, People's Liberation Army General Hospital from January 2011 to December 2014 were retrospectively analyzed. There were 47 cases underwent chimney technique, 6 underwent sandwich technique, 15 underwent fenestration technique and 2 underwent branched stent grafts technique. RESULTS: The average follow-up time was 21 months, range from 3 to 47 months. Completion angiography showed that typeⅠ endoleak of chimney, sandwich, fenestration and branched stent grafts group were 9/47, 5/6, 2/15 and 0, respectively. While at 30-day typeⅠ endoleak reduced to 7/47, 2/6, 0 and 0. During follow-up, there were 3 target vessel stents occlusion in chimney group, the patency rate of target vessel stent was 95.1% (58/61). In sandwich, fenestration and branched stent grafts group, all the target vessel stents kept patent. CONCLUSION: In this study, chimney, sandwich, fenestration and branched stent grafts techniques show good short-term and midterm results, the long-term effects still need further studies.

Association of Resting Heart Rate with Infrarenal Aortic Diameter: A Cross Sectional Study in Chinese Hypertensive Adults.

OBJECTIVE: Elevated resting heart rate (RHR) has been shown to be a risk marker for cardiovascular disease. Results from studies on the effects of RHR in large arteries are limited to the functional changes of those arteries, while the association between RHR and aortic diameter remains largely understudied. METHODS: This was a cross sectional study of hypertensive Chinese adults from rural areas. The maximum infrarenal aortic diameter (maxIAD) from renal arteries to the iliac bifurcation was obtained by ultrasound. MaxIADs in different RHR groups were compared in males and females separately because of the significant differences between sexes. Multiple regression analysis was used to determinate the correlation between RHR and maxIAD. Further interactions between three factors (BMI, smoking, and anti-hypertensive regimens) and RHR for maxIAD were examined using subgroup analysis. RESULTS: 19,200 subjects were enrolled in the study, with an average age of 64.8±7.4 years and 61.6% females. Only 22 cases (0.11%) were detected with AAA, with males (n = 17) presenting a higher AAA incidence than females (n = 5). In subjects ≥65 years, there were 18 (0.19%) AAA, and 15 (83.3%) had a history of smoking. In the total subjects, the mean maxIAD ranged from 15.7±2.1 mm to 15.2±2.2 mm as RHR changed from the lowest quartile to the highest (≥84 bpm) in males, with a similar tendency observed in females. The correlation coefficient of RHR on maxIAD was -0.17 in males and -0.12 in females. Further subgroup analysis revealed that smoking exaggerated the correlation between RHR and maxIAD, but only in females. CONCLUSIONS: A low AAA incidence was observed in this hypertensive Chinese population. There was a negative association between RHR and maxIAD, potentially exaggerated by smoking, especially in females.

Assessing genetic divergence in interspecific hybrids of Aechmea gomosepala and A. recurvata var. recurvata using inflorescence characteristics and sequence-related amplified polymorphism markers.

Conventional hybridization and selection techniques have aided the development of new ornamental crop cultivars. However, little information is available on the genetic divergence of bromeliad hybrids. In the present study, we investigated the genetic variability in interspecific hybrids of Aechmea gomosepala and A. recurvata var. recurvata using inflorescence characteristics and sequence-related amplified polymorphism (SRAP) markers. The morphological analysis showed that the putative hybrids were intermediate between both parental species with respect to inflorescence characteristics. The 16 SRAP primer combinations yield 265 bands, among which 154 (57.72%) were polymorphic. The genetic similarity was an average of 0.59 and ranged from 0.21 to 0.87, indicating moderate genetic divergence among the hybrids. The unweighted pair group method with arithmetic average (UPGMA)-based cluster analysis distinguished the hybrids from their parents with a genetic distance coefficient of 0.54. The cophenetic correlation was 0.93, indicating a good fit between the dendrogram and the original distance matrix. The two-dimensional plot from the principal coordinate analysis showed that the hybrids were intermediately dispersed between both parents, corresponding to the results of the UPGMA cluster and the morphological analysis. These results suggest that SRAP markers could help to identify breeders, characterize F(1) hybrids of bromeliads at an early stage, and expedite genetic improvement of bromeliad cultivars.

In pregnant mice, the infection of Toxoplasma gondii causes the decrease of CD4+CD25+ -regulatory T cells.

Acute maternal infection with Toxoplasma gondii during pregnancy is associated with adverse pregnancy outcomes. Although previous reports have indicated that T. gondii may result in abortion without direct transmission of the parasite to the foetus, the molecular mechanism remains unclear. CD4+CD25+-regulatory T cells are known to be involved in maternal tolerance toward the foetus-bearing alloantigens. With a model of pregnant mice infected with T. gondii, we found that Foxp3 mRNA expression levels in both splenocytes and placenta were reduced markedly during the process of infection. Furthermore, the numbers of splenic CD4+CD25+-regulatory T cells and placental Foxp3+ cells decreased synchronously in the infected mice, and the reduction of splenic CD4+CD25+-regulatory T cells were associated with apoptosis induced by the infection. Additionally, injection of pregnant mice with excretory-secretory antigens (ESA) of T. gondii also resulted in foetal loss, which could be partly prevented by adoptive transfer of CD4+CD25+-regulatory T cells from normal pregnant mice. These data suggest that foetal loss caused by T. gondii can be independent of vertical infection and that the decrease of CD4+CD25+-regulatory T cells during infection may represent a previously unrecognized mechanism for the pathogenesis of abortion caused by this parasite.