Coronavirus infection can cause serious respiratory and digestive system diseases in hu-mans and animals.In recent years,the frequent outbreaks and newly outbreaks of coronavirus in-fection have threatened global public health and the development of livestock and poultry.Howev-er,the development of anti-coronavirus drugs and vaccines was restricted due to the insufficient understanding of the mechanisms of coronavirus pathogenesis and cross species transmission.Re-verse genetic manipulation technology is a powerful tool in virological research,which can be used for the study of pathogenesis mechanisms,replication mechanisms and function analysis of protein.It is also be used in the development of attenuated or gene labeled vaccines,and antiviral drugs.Due to the large genome and complex structure of the coronavirus,the reverse genetic manipulation technology of coronavirus has been lagging for a long time.With the continuous updating of molec-ular biology methods,various new construction strategies have emerged.This article focuses on the construction strategy of the reverse genetic operating system for coronavirus,as well as its applica-tion in virus transmission and pathogenic mechanisms,and development of vaccines,which will provide favorable tools for the prevention and control of the coronavirus infection.

Objective:To identify the differentially expressed long-chain non-coding RNA(lncRNA) and mRNA using ribonucleic acid sequencing(RNA-seq), and construct a competing endogenous RNA(ceRNA) regulatory network in mice with sepsis-associated encephalopathy.Methods:Ten clean-grade healthy male C57BL/6 mice, aged 6-8 weeks, weighing 20-25 g, were divided into 2 groups( n=5 each) using a random number table method: sham operation group(group Sham) and sepsis group(group Sepsis). Sepsis was induced by cecal ligation and puncture(CLP) in group Sepsis, while group Sham only underwent laparotomy without CLP. Morris water maze test and contextual fear conditioning test were performed to detect the cognitive function on 1 day before CLP and 3 days after CLP. Three mice were randomly sacrificed in group Sham, and 3 mice with the worst results in the cognitive function test were sacrificed in group Sepsis. The hippocampal tissues were obtained for RNA-seq via the BGISEQ-500 platform, and the differentially expressed mRNA and lncRNA were identified. The differentially expressed mRNAs and lncRNAs were visualized and analyzed by Dr. Tom platform provided by Shenzhen BGI Technology Service Co., Ltd., and the ceRNA regulatory network was constructed using the online visualization tool Cytoscape software. Results:Compared with group Sham, the escape latency was significantly prolonged, and the percentage of time of staying at the target quadrants and percentage of time spent freezing were decreased in group Sepsis( P<0.05). A total of 62 differentially expressed lncRNAs were obtained from RNA-seq, of which the expression of 45 lncRNAs was up-regulated and the expression of 17 lncRNAs was down-regulated.There were 282 differentially expressed mRNAs identified from RNA-seq, of which the expression of 173 mRNAs was up-regulated, and the expression of 109 mRNAs was down-regulated.Gene Ontology enrichment analysis revealed that the differentially expressed mRNAs were involved in biological processes such as memory, learning or memory, inflammatory responses, regulation of aging-related behavioral decline, and regulation of synaptic plasticity. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that differentially expressed mRNAs were enriched in IL-17 signaling pathway, TNF signaling pathway, NF-κB signaling pathway and etc. KDA analysis was performed on the differentially expressed mRNAs to identify the key driver genes, and the results showed that Ch25h, Il6ra, Lcn2, Sgk1, Nr4a3, Osm, Saa3, Ccl7, Sqle, Dhcr24 were the key SAE genes.A competing endogenous RNA regulatory network was successfully constructed based on 9 lncRNAs, 28 mRNAs and 134 miRNAs in the hippocampus of mice with SAE. Conclusions:The results of RNA-seq find that 10 mRNAs including Ch25h, Il6ra, Lcn2, Sgk1, Nr4a3, Osm, Saa3, Ccl7, Sqle, Dhcr24 and lncRNAs such as Rian, Gm35874 and Gm34347 are key genes regulating SAE in mice. Meanwhile, a ceRNA regulatory network based on lncRNA-miRNA-mRNA is successfully constructed in the hippocampus of mice with SAE.

Objective:To investigate the differential expression profile of circular RNA (circRNA) in high glucose-cultured human retinal vascular endothelial cells (HRVECs).Methods:HRVECs were divided into high glucose group, normal control group and hypertonic control group, and were cultured in 25 mmol/L glucose medium, 5.5 mmol/L glucose medium and 19.5 mmol/L mannitol+ 5.5 mmol/L glucose medium for 24 hours accordingly.The differentially expressed circRNA molecules between high glucose group and normal control group were screened by circRNA microarray analysis.The expression of the most significant differentially expressed circRNAs in different groups was verified by real-time quantitative PCR.The possible microRNA (miRNA) targets were analyzed through the Circular RNA Interactome database.Results:It was found that 448 circRNAs were differentially expressed (FC≥1.5 or FC≤0.67, P<0.05) in high glucose-cultured HRVECs, among which 182 were up-regulated and 266 were down-regulated.The top 3 significantly up-regulated circRNAs were hsa_circ_0002938, hsa_circ_0008036, and hsa_circ_0001946, and the top 3 significantly down-regulated circRNAs were hsa_circ_0035277, hsa_circ_0008344, and hsa_circ_0001874.Compared with normal control group and hypertonic control group, the relative expressions of top 3 up-regulated circRNAs were significantly enhanced and the relative expressions of top 3 down-regulated circRNAs were significantly reduced in high glucose group, showing statistically significant differences (all at P<0.05).No significant difference was found in the differentially expressed circRNAs between normal control group and hypertonic control group (all at P>0.05). Conclusions:CircRNAs are differentially expressed in high glucose-cultured HRVECs, and the differentially expressed circRNAs may be involved in the regulatory mechanism of diabetic retinopathy.

Objective To evaluate the role of spinal kinesin superfamily motor protein 17 (KIF17)in remifentanil-induced hyperalgesia in rats with incisional pain. Methods Forty male Sprague-Dawley rats, aged 2-3 months, weighing 240-260 g, were divided into 5 groups(n=8 each)using a random number table: control group(group C), remifentanil group(group R), incisional pain group (group I), remifentanil plus incisional pain group(group R+I)and remifentanil plus incisional pain plus KIF17 inhibitor group(group R+I+M). Normal saline 15 ml was intravenously infused for 60 min in group C. Remifentanil 1 μg·kg-1·min-1was intravenously infused for 60 min in group R. In group I, in-cisional pain model was established, and normal saline 15 ml was intravenously infused for 60 min at the same time. In group R+I, incisional pain model was established, and remifentanil 15 ml was intravenous-ly infused for 60 min at the same time. In group R+I+M, KIF17 inhibitor Myr-Rc-13 10 μg(in 10 μl dimethyl sulfoxide)was injected intrathecally, and remifentanil 1 μg·kg-1·min-1was intravenously in-fused for 60 min while incisional pain model was established. The mechanical paw withdrawal threshold (MWT)and thermal paw withdrawal latency(TWL)were measured at 24 h before remifentanil or normal saline infusion and at 2, 6, 24 and 48 h after the end of infusion. The rats were sacrificed after the last measurement of pain threshold, and the lumbar segment of the spinal cord was removed for determination of the expression of KIF17 and phosphorylated KIF17(pKIF17)by Western blot. Results Compared with group C, MWT was significantly decreased, TWL was shortened, and the expression of KIF17 and pKIF17 was up-regulated in R, I and R+I groups(P<005). Compared with R and I groups, MWT was signifi-cantly decreased, TWL was shortened, and the expression of KIF17 and pKIF17 was up-regulated in group R+I(P<005). Compared with group I+R, MWT was significantly increased, TWL was prolonged, and the expression of KIF17 and pKIF17 was down-regulated in group R+I+M(P<005). Conclusion In-creased activity of KIF17 is involved in the development and maintenance of remifentanil-induced hyperalge-sia in rats with incisional pain.

Objective To determine the relative and independent risk factors of survival in patients with non-hepatitis B and non-hepatitis C hepatocellular carcinoma (NBNC-HCC).Methods The clinical records of 109 patients who underwent surgical resection for NBNC-HCC at Tianjin Medical University Cancer Institute & Hospital between January 2010 and January 2013 were retrospectively analyzed.The risk factors influencing disease-free survival (DFS) and overall survival (OS) were used as primary outcome measures.Univariate analysis was conducted to determine the relative risk factor predicting prognosis of NBNC-HCC,and the Cox proportional hazards model was used to determine independent risk factors of DFS and OS.Results For the 109 NBNC-HCC patients,the 1-,2-,3-year overall survival rates were 90.8％,78.0％ and 65.1％,respectively.The compounding disease-free survival rates were 74.0％,63.3％ and 55.8％,respectively.Univariate analysis showed the AFP level,ascites,and TNM staging were the risk factors of OS (all P ＜ O.05).The AFP level,ascites,BCLC stage,TNM staging were related with DFS (all P ＜ 0.05).Multivariate analysis demonstrated AFP and ascites to be the independent risk factors of OS and DFS.Conclusions AFP and ascites were independent risk factors of OS and DFS.For the NBNC-HCC patients,a strong positive AFP with ascites indicated poor prognosis.

Objective To evaluate the effect of 6% hydroxyethyl starch 130∕0. 4 on acute kidney injury in elderly patients in a prospective, multicenter, randomized, double?blind, controlled clinical tri?al. Methods A total of 120 patients of both sexes, aged 65-82 yr, weighing 56-83 kg, of American So?ciety of Anesthesiologists physical status Ⅰ or Ⅱ, scheduled for elective orthopaedics and hernia surgery, were divided into either hydroxyethyl starch group ( group HES) or lactated Ringer′s solution group ( group LR) , with 60 patients in each group. Hydroxyethyl starch and lactated Ringer′s solution were infused intra?venously at a rate of 7. 5 ml∕kg during 1st hour of surgery in HES and LR groups, respectively. Lactated Ringer′s solution was then infused at a rate of 5 ml∕kg starting from 2nd hour of surgery until the end of sur?gery in both groups. Before surgery, at the end of surgery and at 1, 3 and 5 days after surgery, blood sam?ples and urine specimens were collected for determination of the concentrations of neutrophil gelatinase?asso?ciated lipocalin, interleukin?18 (IL?18), plasma creatinine, urine β2 microglobulin and urine albumin.
The estimated glomerular filtration rate was calculated. Results The level of urine IL?18 was significantly higher at each time point after surgery than before surgery and immediately after the end of surgery ( P<0.05) . There were no significant differences between the two groups in the levels of urine IL?18, plasma creatinine, plasma and urine neutrophil gelatinase?associated lipocalin, plasma IL?18, urineβ2 microglob?ulin and urine albumin and estimated glomerular filtration rate at each time point ( P>0.05) . Conclusion Compared with lactated Ringer′s solution, 6% hydroxyethyl starch 130∕0.4 does not aggravate acute kidney injury in elderly patients.

Objective To evaluate the relationship between extracelluar signal?regulated protein kinase 1∕2 (ERK1∕2) and signal transducer and activator of transcription 3 (STAT3) signaling pathways involving in cardioprotection induced by diazoxide postconditioning in rats. Methods Sixty adult male Sprague?Dawley rats, aged 3 months, weighing 240-260 g, were randomly divided into 5 groups ( n=12 each) using a random number table: sham operation group ( SH group ) , ischemia?reperfusion ( I∕R ) group, diazoxide postconditioning group ( D group ) , ERK1∕2 inhibitor U0126 group ( U group ) , and STAT3 inhibitor Stattic group ( St group) . Myocardial I∕R was produced by occlusion of anterior descending branch of the coronary artery followed by 120 min reperfusionIn I∕R and D groups, 0?4% dimethyl sulfoxide 1 ml and 7 mg∕kg diazoxide ( in 1 ml of 0?4% dimethyl sulfoxide) was injected through the femoral vein at the onset of reperfusionIn U and St groups, U0126 100 μg∕kg and Stattic 500 μg∕kg were injected through the femoral vein at 10 min before reperfusion, and the other procedures were similar to those previously described in group DAt 120 min of reperfusion, the rats were sacrificed, and myocardial specimens were obtained from the left ventricle for determination of myocardial infarct size, cell apoptosis, and ERK1, ERK2 and STAT3 mRNA expression ( real?time PCR), and phosphorylated ERK1∕2 ( p?ERK1∕2) and phosphorylated STAT3 (p?STAT3) (using Western blot). Apoptosis index (AI) was calculated. Results Compared with group S, the myocardial infarct size and AI were significantly increased, and the expression of ERK1, ERK2 and STAT3 mRNA, p?ERK1∕2 and p?STAT3 was down?regulated in group I∕R. Compared with group I∕R, the myocardial infarct size and AI were significantly decreased, and the expression of ERK1, ERK2 and STAT3 mRNA, p?ERK1∕2 and p?STAT3 was up?regulated in group D. Compared with group D, the myocardial infarct size and AI were significantly increased in U and S groups, the expression of ERK1, ERK2 and STAT3 mRNA, p?ERK1∕2 and p?STAT3 was down?regulated in group U, and the expression of STAT3 mRNA and p?STAT3 was down?regulated, and no significant change was found in ERK1 and ERK2 mRNA and p?ERK1∕2 expression in group S. Conclusion STAT3 signaling pathway is located downstream of ERK1∕2 signaling pathway in the mechanism by which diazoxide postconditioning reduces myocardial I∕R injury in rats.

Objective To evaluate the effects of a single IV lidocaine bolus dose on the minimal alveolar concentration (MAC)of sevoflurane. Methods Patients (n=90), aged 25-65 years whose Anesthesiologists (ASA) classification wasⅠorⅡand underwent elective surgery on trunk under general anesthesia, were randomly divided into 3 groups with 30 cases in each group:high-dose lidocaine group (group H), low-dose lidocaine group (group L) and control group (group C). They were induced by sevoflurane inhalation, and ventilated by LMA (laryngeal mask airway). After a 15 minutes equilibration period with the above sevoflurane concentration , the medication to be studied (2%lidocaine 1.5 mg/kg for group H , 2%lidocaine 0.75 mg/kg for group L, 0.9%saline 5mL for group C) was administered for 3 minutes before the skin incision. The response to skin incision (movement versus no movement) was recorded in the first minute after skin incision. The MAC for sevoflu?rane was determined using the Dixon′s up and down method. Values of mean arterial pressure (MAP), heart rate (HR), and BIS were recorded at 1 minute and 5 minutes after being monitored (average values were noted as T0), immediately before the administration of medication (T1), immediately before the skin incision (T2) and 1 minute after the skin incision(T3). Results MAC in group H (2.00%± 0.17%) was lower than that in group C (2.22%± 0.18%) by approximately 0.22%,and which was lower than that of group L ( 2.21%± 0.14%) by approximately 0.21%(F=7.054,P<0.05). No significant differ?ence in the MAC of sevoflurane was noted between group L and group C. The values of HR, MAP and BIS all decreased at T 2 and increased at T3 in all 3 groups (all P<0.05). No significant difference in HR, MAP or BIS was observed between T0 and T1 in all three groups. The values of HR and BIS were lower in group H than those in group C and group L at T2 and T3. The values of MAP were lower in group L and group H than those in group C at T2 and T3. The value of MAP were lower in group H than that in group L at T2(all P<0.05). Conclusion A singleⅣ1.5 mg/kg lidocaine decreases MAC of sevoflurane, but the decreased amplitude (11%) does not reach expectation.

OBJECTIVE: To evaluate the efficacy of sublingual immunotherapy (SLIT) with Dermatophagoides farinae drops for allergic rhinitis (AR) of different symptom severity. METHOD: This retrospective analysis to receive SLIT treatment of 143 cases of patients with allergic rhinitis, according to the severity of disease symptoms divid- ed into two groups, moderate group (62 patients) and severe group (81 patients). Before SLIT and after SLIT for half year, 1 year and 1. 5-2.0 years, the TNSS, TMS and sign scores of patients with allergic rhinitis were evaluated. RESULT: The TNSS, TMS and sign scores had continuously improved significantly after SLIT for half year, 1 year and 1.5-2.0 years in two groups as compared with baseline (P < 0.05). Before SLIT, TNSS and sign scores of severe group had a significantly higher level than moderate group (Z = 10.40, 2.40, P < 0.05), while TMS of two groups had no significant differences (Z = 0.00, P > 0.05). Half year after SLIT treatment, in two groups for sign scores, there were significant differences (Z = 3.32, P < 0.05), and there were no significant differences for TNSS (Z = 1.58, P > 0.05) and TMS (Z = 0.37, P > 0.05). 1 and 1.5-2.0 years after SLIT, there were no significant differences in two groups for TNSS, TMS and sign scores (P > 0.05). CONCLUSION SLIT with Dermatophagoides farinae drops for 1.5-2.0 years is effective in the patients with allergic rhinitis of different symptom severity. And equivalent efficacy could be achieved for different symptom severity.
Administration, Sublingual ; Animals ; Antigens, Dermatophagoides ; administration & dosage ; Dermatophagoides farinae ; Humans ; Retrospective Studies ; Rhinitis, Allergic, Perennial ; drug therapy ; Sublingual Immunotherapy ; Treatment Outcome

Objective To evaluate the role of signal transducer and activator of transcription 3 (STAT3) signaling pathway in the reduction of myocardial ischemia-reperfusion (I/R) injury by diazoxide postconditioning in rats.Methods Sixty adult male Sprague-Dawley rats,aged 3 months,weighing 240-260 g,were randomly divided into 5 groups (n =12 each):sham operation group (S group),I/R group,vehicle group (V group),diazoxide postconditioning group (D group),and STAT3 signaling pathway inhibitor Stattic group (St group).Myocardial I/R was produced by 30 min occlusion of left anterior descending branch of coronary artery followed by 120 min reperfusion.In V and D groups,0.4％ dimethyl sulfoxide and 7 mg/kg diazoxide (in 1 ml of 0.4％ dimethyl sulfoxide) were injected through the femoral vein at the onset of reperfnsion,respetively.In St group,Stattic was injected through the femoral vein 10 min before reperfusion,and the other procedures were the same as those in D group.The infarct size (IS) and myocardial apoptosis were detected by TTC staining and TUNEL,respectively.Apoptotic index (AI) was calculated.STAT3 mRNA expression in myocardial tissues was detected using RT-PCR.Western blot was used to detect the phosphorylation of STAT3.Results Compared with S group,the IS and AI were significantly increased and the expression of STAT3 mRNA and phosphorylation of STAT3 were decreased in I/R group (P ＜ 0.05).Compared with I/R group,the IS and AI were significantly decreased and the expression of STAT3 mRNA and phosphorylation of STAT3 were increased in D group (P ＜ 0.05).There was no significant difference in IS,AI,expression of STAT3 mRNA and phosphorylation of STAT3 between V group and St group (P ＞0.05).Compared with group D,the IS and AI were significantly increased and the expression of STAT3 mRNA and phosphorylation of STAT3 were decreased in St group (P ＜ 0.05).Conclusion STAT3 signaling pathway is involved in the reduction of myocardial I/R injury by diazoxide postconditioning in rats.