Objective To evaluate the effects of lentivirus-delivered short hairpin RNA (shRNA) targeting human papillomavirus 16 (HPV16) E7 gene on the expression of 4 kinds of DNA methyltransferases (DNMTs),including DNMT1,DNMT3A,DNMT3B and DNMT3L,in HPV16-positive cervical cancer cell line SiHa.Methods The recombinant plasmid containing HPV16 E7 gene-targeting shRNA was constructed firstly.Then,the BLOCK-iTTM lentiviral RNAi expression system kit was used to package the lentiviral vector,which was transfected into 293T cells.The lentivirus-containing supernatants were collected at 48 and 72 hours after transfection.The SiHa cells were divided into 3 groups to be cultured with lentiviral supernatant containing HPV16 E7 gene-targeting shRNA recombinant plasmids mixed with complete medium at a ratio of 1:1 (shRNA group),lentiviral supernatant containing empty plasmids mixed with complete medium at a ratio of 1:1 (negative control group),and complete medium alone (blank control group),respectively.Real-time fluorescence-based quantitative PCR (qRT-PCR) was performed to measure mRNA expression of HPV16 E7 and 4 kinds of DNMTs in the above 3 groups at 0,48,96 hours after infection,and Western blot analysis to determine protein expression of the 4 DNMTs at 48,96 hours after infection.Results There were no significant differences in the mRNA expression of HPV16 E7 and the 4 DNMTs among the shRNA group,negative control group and blank control group at 0 hour after infection (all P ＞ 0.05).At 48,96 hours after infection,the mRNA expression of HPV16 E7 and the 4 DNMTs decreased significantly in the shRNA group compared with the negative control group and blank control group (all P ＜ 0.05),but did not differ between the negative control group and blank control group (all P ＞ 0.05).Additionally,E7,DNMT1,DNMT3A,DNMT3B and DNMT3L gene-silencing efficiencies in the shRNA group were 71.13％,50.53％,13.72％,46.27％ and 17.92％ at 48 hours,and 83.50％,74.2％,47.8％,64.7％ and 48.9％ at 96 hours after infection,respectively.Western blot analysis showed that the protein expression of the 4 DNMTs significantly decreased in the shRNA group compared with the negative control group and blank control group at 48,96 hours after infection (all P ＜ 0.01).Moreover,the protein expression of DNMT1,DNMT3A,DNMT3B and DNMT3L in the shRNA group gradually decreased over time,and was inhibited by 84％,37.2％,59.8％ and 49.3％ at 48 hours respectively,and by 73.1％,68.7％,55.5％ and 65.5％ at 96 hours after infection respectively.Conclusion Targeted silencing of E7 gene in HPV16-positive SiHa cells can interfere with the mRNA and protein expression of DNMT1,DNMT3A,DNMT3B and DNMT3L.

Objective To investigate the brain metabolic changes and evaluate their spatial distributions after traumatic axonal injury (TAI)in rats by using proton magnetic resonance spectroscopy(1H-MRS).Methods The TAI model was made by subjecting the head of the rats to the linear and angular accelerations.The multi-voxel MRS was employed to detect the tissue metabolic state at the levels of hippocampus-caudate and pons prior to injury and at 24 hours after injury.The alterations of NAA/Cr,NAA/Cho and Cho/Cr values as well as the spatial distribution of NAA/Cr reduction were accessed. Immunohistochemical staining for β-APP was used to observe the injured axons. Results A siguificantdecrease in NAA/Cr and NAA/Cho(P<0.05)and subtle increase in Cho/Cr(P>0.05)were observed in rats at 24 hours after TAI in comparison to the pre-injury levels.Notable decrease in NAA/Cr value was observed in the areas including the brain stem,hippocampus,internal capsule,corpus callosum and thalamus,where axonal injuries were confirmed by the histological examination. Conclusion Metabolic imbalances Occur in the brains of rats with TAI.with notable changes in the brain stem and the hippocampus.

Objective To observe the spatiotemporal characteristics of the micro-structural injury in a rat model of diffuse axonal injury (DAI) and quantitatively assess the axonal injury severity in the vulnerable areas. Methods The 7.0 T MRI was performed in rats in DAI group (n =20) and control group ( n = 15 ) to synthesize the diffusion tensor imaging ( DTI) parameter map and calculate the parameter value of the vulnerable areas. Immunohistochemistry was used to detect β-APP expression in the vulnerable areas and the IPP software to quantitatively assess the axonal injury severity. Results Compared with the control group, FA and AD maps showed local signal defection or reduction in the corpus callosum and their values decreased significantly in the brain stem and corpus callosum in the DAI group (P ＜0.01 ). The integrated optical density (IOD) value of the vulnerable areas in the DAI group was significantly higher than that of the control group ( P ＜ 0. 01 ) , with the highest level in the brain stem (P＜0.05). The normalized FA, AD and ADC in the vulnerable areas were correlated negatively with the IOD (P ＜ 0.05). Conclusion DTI can detect invisible micro-structural injury in the vulnerable areas and quantitatively assess the axonal injury severity in vivo in the early stage.

Objective To investigate whether HSP90α could be a sensitive and specific serum biomarker for the diagnosis and progression of lung cancer. Methods In the present study, different secretomic analy-ses on the two human lung adenocarcinoma cell lines CL1-0 and CL1-5 with low and high metastatic poten-tial, respectively, were performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ma-trix-assisted laser desorption/ionization time-of-flight mass spectrometry. The candidate biomarker was con-firmed by Western blotting, and was further analyzed in 224 serum samples including 141 lung cancer, 37 benign pulmonary diseases, as well as 46 healthy individuals using ELISA assay. Results HSP90α was sig-nificantly upregulated in the CM of CL1-5 cells. It was found that the levels of HSP90α were specifically ele-vated in the sera of non-small cell lung cancer compared with other groups. At the cut-off point 0.535 on the receiver operating oharacteristie curve, HSP90α could comparatively discriminate lung cancer from benign lung disease and healthy control groups with sensitivity of 0. 817, specificity 0. 919 and total accuracy 80. 14%. Conclusion HSP90α may be a potential useful serum biomarker for discriminating lung cancer from benign lung diseases and healthy individuals and staging of non-small cell lung cancer.

Objective To investigate the early alterations of neurometabolites in the splenium of corpus callosum by proton magnetic resonance spectroscopy (~1H-MRS) in patients with diffuse axonal injury (DAI) at early stage and their prognostic value. Methods Twenty-one patients with DAI underwent 1H-MRS examination in the splenium of corpus callosum 2 to 14 d after injury, the neurometabolite alterations were evaluated and compared with those of 10 healthy subjects (normal controls). Logistic regression analysis was performed to explore the correlation among neurometabolite alterations, clinical indexes and Glasgow outcome scale (GOS) six months after injury. Results There was a significant decrease in NAA/Cr and NAA/Cho and increase in Cho/Cr in the splenium of corpus callosum in patients with DAI as compared with control group (P<0.05), and these trends tended to be more evident with the increase of injury severity. NAA/Cr and NAA/Cho in patients with poor outcomes were lower than those in patients with good outcomes (P<0.05). NAA/Cr, NAA/Cho and Cho/Cr predicted long-term outcome with 89% accuracy, and the combination with GOS provided the predictive accuracy of 94%. Conclusion ~1H-MRS examination in the splenium of corpus callosum at early stage of DAI can depict brain injury severity, and is useful in predicting outcomes.

Objective To compare the effect of 89Sr radionuclide therapy and radiotherapy in the treatment of metastatic cancer pain of vertebral column. Methods 80 patients with cancer pain of vertebral metastasis in the second People's hospital of Yibin from April 2015 to April 2017, were randomly divided into the control group treated by radiotherapy treatment (n=40) and the the observation group treated by 89Sr radionuclide therapy(n=40), and the effect of treatment were compared between two groups. Results In the observation group, the total effective rate of pain response in patients with metastatic cancer pain of vertebral column was 85.0％, and the control group was 82.5％. There was no significant difference between two groups. The onset time of treatment in the observation group was (6.5±1.7)d, significantly shorter than that of the control group (12.9±2.6)d, and the difference was statistically significance (P<0.05). Conclusion The application of 89Sr radionuclide therapy in the treatment of vertebral metastatic pain is equivalent to the radiotherapy in improving the pain response, but the efficacy could be achieved in a short period of time, so it is worth popularizing widely in clinical practice.

Objective To discuss the clinical application of reverse dorsal metatarsal artery perforator flap for reconstruction of forefoot soft tissue defects.Methods Since January,2008 to March,2016,43 patients with forefoot soft tissue defect due to various causes were reconstructed with the flap based on dorsal metatarsal artery perforator.Dorsal pedis was used as a donor site with dorsal metatarsal artery perforator as the donor artery.The flap size varied from 2.0 cm × 4.0 cm to 6.0 cm × 8.0 cm.Among them,27,8,4 and 4 cases were reconstructed with flap based on first,second,third and fourth dorsal metatarsal artery perforator flap respectively.Results All the patients were followed-up which ranged from 3 months to 24 months.Six flaps suffered from post operative venous congestion.Out of 6,4 survived with early emergency management while the remaining 2 suffered epidermal necrosis which survived with regular dressing change.Eventually,all the flaps survived.They had good texture and they were elastic,good-looking and very wear-resisting.Walking function was normal.Conclusion Reverse dorsal metatarsal artery perforator flap is an ideal choice in reconstruction of small to medium sized soft tissue defects of forefoot.

Objective To determine the serum soluble CD226/PTA1(sCD226/PTA1)level in pa-tients with psoriasis vulgaris.Methods Serum sCD226/PTA1level was measured by sandwich ELISA in30patients with psoriasis vulgaris before treatment(17patients in active stage and13in inactive stage),10patients after treatment,and15healthy individuals as a control.Results Serum sCD226/PTA1level was significantly increased in patients with psoriasis compared with that in healthy controls(P

Objective:To systematically review the severe risk in common chronic diseases and coronavirus disease 2019 (COVID-19) cases.Methods:PubMed, Embase, Cochrane Library, China National Knowledge Infrastructure, Wanfang Database, China Biology Medicine disc, medRxiv, SSRN and ChinaXiv were searched for clinical and epidemiological studies that reported chronic diseases in patients with COVID-19. Only studies of severe COVID-19 in comparison with non-severe controls were included. The prevalence rates of chronic diseases including chronic obstructive pulmonary disease (COPD), diabetes mellitus, hypertension, malignant tumor, cardiovascular diseases, cerebrovascular disease, chronic kidney disease, and chronic liver disease were estimated. Pooled odds ratio ( OR) with 95% confidence interval ( CI) between patients with severe COVID-19 and non-severe groups were calculated. R 3.6.3 software was used for meta-analysis. Results:The search yielded 2 455 articles. A total of 19 eligible comparative studies with 4 792 patients were included in a quantitative analysis. Meta-analysis showed that there was a proportion of 55.0% (95% CI 40.0%-80.0%) male among patients with COVID-19, and the overall pooled prevalence of any chronic diseases in COVID-19 cases was 30.4% (95% CI 24.0%-37.0%). The most prevalent comorbidity was hypertension (16.9%(95% CI 14.0%-20.0%)), followed by diabetes mellitus (8.3%(95% CI 8.0%-9.0%)). The proportion of male patients with severe COVID-19 was higher than that of male patients with non-severe COVID-19 (64.4% vs 52.8%, OR=1.49, 95% CI 1.08-2.05, Z=4.63, P<0.01). The prevalence rates of COPD, cerebrovascular disease, diabetes mellitus, chronic kidney disease, hypertension, cardiovascular diseases and malignant tumor in severe COVID-19 patients were higher than those of non-severe patients ( OR=5.77, 95% CI 3.80-8.74; OR=4.47, 95% CI 2.71-7.38; OR=3.55, 95% CI 2.86-4.40; OR=3.05, 95% CI=1.76-5.28; OR=2.82, 95% CI=1.96-3.97; OR=2.39, 95% CI=1.77-3.23; OR=2.15, 95% CI 1.27-3.66, respectively, Z=8.37, 6.01, 11.60, 4.20, 5.46, 5.71, 3.12, all P<0.01). There was no significant difference in the prevalence of chronic liver disease between severe and non-severe patients ( OR=1.35, 95% CI 0.84-2.17, P=0.11). Conclusion:COVID-19 patients with chronic diseases have higher risk of developing severe disease, and the ORs from high to low are COPD, cerebrovascular disease, diabetes mellitus, chronic kidney disease, hypertension, cardiovascular diseases and malignant tumor.

Objective:To investigate the efficacy of 3D-printed quantitative bone implants assisting second-stage Masquelet technique for the treatment of long-segment bone defect following Gustilo type IIIB and IIIC tibial fractures.Methods:A retrospective case series analysis was made on 26 patients with long-segment bone defect following Gustilo type IIIB and IIIC tibial fractures treated in Wuxi Ninth People′s Hospital from July 2015 to December 2020, including 20 males and 6 females; aged 19-63 years [(46.5±4.5)years]. Gustilo classification was type IIIB in 23 patients and type IIIC in 3. In the first stage, all patients had thoroughly emergent debridement, removal of all free bone pieces, restoration of the length and force line plus externally fixion, and vacuum sealing drainage (VSD) of the residual wound. After 2-7 days, the external fixation was removed and replaced by internal fixation, with the bone cement filling in the defect area and the free flap covering the wound. The length of tibial bone defect was 5-14 cm [(6.3±0.4)cm], and the tibial defect volume was 12.2-73.1 cm 3 [(33.6±9.2)cm 3]. In the second stage (6-19 weeks after injury), the bone cement was removed, followed by autologous bone grafting. Prior to bone grafting, digital technology was used to accurately calculate the bone defect volume, and an equal volume of bone harvesting area was designe to produce the 3D printed osteotomy template. Bone grafting was conducted after bone removal according to the osteotomy template during operation. The success rate of one-time iliac bone extraction, bone harvesting time, and bleeding volume were recorded. Pain in the bone extraction area was evaluated by visual analogue score (VAS) at 1 day and 1 month after operation and at the last follow-up. Wound healing, complications, and bone healing were observed. Life quality was evaluated by health survey brief form (SF-36) including scores of physical component summary (PCS) and mental component summary (MCS) before bone grafting and at the last follow-up. Results:All the patients were followed up for 13-53 months [(32.3±12.5)months]. One-time iliac bone extraction was successful in all the patients. Bone harvesting time was 15-30 minutes [(21.0±2.5)minutes]. The bleeding volume was 50-120 ml [(62.3±29.0)ml]. The VAS was 1-4 points [(1.2±0.9)points] at 1 day after operation, higher than these (0.0±0.0)points at 1 month after operation and at the last follow-up (all P<0.01). Totally, 25 patients obtained wound healing after operation, except for 1 patient with superficial wound infection after bone grafting that was healed by dressing change. There was 1 patient with bone infection after 3 months of bone grafting that was healed by repeated surgery with Masquelet technique in the first and second stage. Besides, 2 patients had symptoms of cutaneous nerve injury in the iliac donor area. The time of bone healing was 4-7 months [(5.8±0.8)months]. The scores of PCS and MCS in SF-36 at the last follow-up were (73.6±12.8)points and (83.6±13.2)points, significantly higher than those before bone grafting [(46.8±0.5)points, (60.7±2.0)points] (all P<0.01). Conclusion:Second-stage Masquelet technique with 3D printed quantitative bone implants for the treatment of long-segment bone defect following Gustilo type IIIB and IIIC tibial fractures is associated with shortened bone harvesting time, attenuated pain, reduced complications, accelerated bone healing and improved function.