Objective Genotypes(G)1,3,and 5 of the Japanese encephalitis virus(JEV)have been isolated in China,but the dominant genotype circulating in Chinese coastal areas remains unknown.We searched for G5 JEV-infected cases and attempted to elucidate which JEV genotype was most closely related to human Japanese encephalitis(JE)in the coastal provinces of China. Methods In this study,we collected serum specimens from patients with JE in three coastal provinces of China(Guangdong,Zhejiang,and Shandong)from 2018 to 2020 and conducted JEV cross-neutralization tests against G1,G3,and G5. Results Acute serum specimens from clinically reported JE cases were obtained for laboratory confirmation from hospitals in Shandong(92 patients),Zhejiang(192 patients),and Guangdong(77 patients),China,from 2018 to 2020.Seventy of the 361 serum specimens were laboratory-confirmed to be infected with JEV.Two cases were confirmed to be infected with G1 JEV,32 with G3 JEV,and two with G5 JEV. Conclusion G3 was the primary infection genotype among JE cases with a definite infection genotype,and the infection caused by G5 JEV was confirmed serologically in China.

Objective:Based on the specific cleavage and non-specific "trans-cleavage" activities of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein(CRISPR/Cas13), we established a visually amplification-free rapid detection technique of 2019-nCoV nucleic acid. This technique is easily processed with a low detection limit and good specificity.Methods:According to the 2019-nCoV gene sequence, specific CRISPR RNAs were screened and designed by bioinformatics analysis, and then synthesized as universal signal-strained RNA transcription targets in vitro to establish and optimize the reaction system. Moreover, the 2019-nCoV pseudoviral nucleic acid was used as a standard substance to evaluate the detection limit. A total of 65 positive samples were collected from various 2019-nCoV variants, while 48 negative samples included other clinically common respiratory pathogens, such as influenza A virus, influenza B virus, human parainfluenza virus, Klebsiella pneumonia, etc. All samples were tested by quantitative PCR (qPCR), digital PCR, and the method established in this study. The sensitivity and specificity of the newly established method were analyzed and evaluated. Results:With the newly established technique, the detection time for 2019-nCoV nucleic acid could be minimized to 6 minutes. In addition, the detection limit was 14 copies/μl when assisted by the displaying instrument, whereas it increased to 28 copies/μl with the naked eye. This technique had a sensitivity and specificity of 98.5% (66/67) and 100% (46/46) respectively, showing no statistically significant difference compared to the gold standard qPCR( P=1). Conclusions:This study has successfully established a CRISPR/Cas13a-based visually rapid detection technique for 2019-nCoV nucleic acid. This technique offers the advantages of a simple process, convenient operation, low environmental operating requirements, a detection limit close to qPCR, and a strong potential for on-site testing applications.

Objective To compare image quality of 5.0T and 3.0T non-contrast MRI for displaying insulinoma.Methods Twelve patients with insulinoma were prospectively enrolled,and non-contrast abdominal T1WI,T2WI as well as diffusion-weighted imaging(DWI)were acquired using 5.0T and 3.0T MR scanners,respectively.The subjective scores of image quality of each sequence of 5.0T and 3.0T MRI,also of tumor-pancreas parenchyma contrast scores were compared.The signal-to-noise ratio(SNR)and contrast-to-noise ratio(CNR)of insulinomas were observed,and the displayed rate of insulinoma by each sequence and overall MRI were compared.Results The subjective scores of 5.0T T1WI and DWI were higher than those of 3.0T T1WI and DWI(both P＜0.05),but not significantly different between 5.0T and 3.0T T2WI(P=0.166).Furthermore,the tumor-pancreas parenchyma contrast score of 5.0T T1WI was higher than that of 3.0T T1WI(P=0.023),but not significantly different between 5.0T and 3.0T T2WI,nor between 5.0T and 3.0T DWI(both P＞0.05).SNR of insulinomas on 5.0T T2WI were higher than on 3.0T T2WI(P=0.015),however,no significant difference of SNR was found between 5.0T and 3.0T T1WI,nor between 5.0T and 3.0T DWI(both P＞0.05).CNR of insulinomas on all 5.0T MRI were not significantly different with those on 3.0T MRI(all P＞0.05).The displayed rate of insulinoma on 5.0T T1WI,T2WI and DWI was 100％(12/12),66.67％(8/12)and 83.33％(10/12),respectively,on 3.0TT1WI,T2WI and DWI was 75.00％(9/12),58.33％(7/12),66.67％(8/12),respectively.The overall displayed rate of insulinoma on 5.0T and 3.0T MRI was 100％(12/12)and 83.33％(10/12),respectively.Conclusion Compared with 3.0T MRI,5.0T MRI was superior for displaying insulinoma,hence being helpful for diagnosis.

Objective To investigate the influencing factors for white matter hyperintensity （WMH） in middle-aged and elderly patients with hypertension， and to establish and verity a nomogram prediction model. Methods A total of 198 middle-aged and elderly patients with hypertension and WMH who were hospitalized in our hospital from January 2022 to April 2023 were enrolled. Related clinical data were analyzed， and related data were recorded. A binary logistic regression analysis was used to investigate the independent risk factors for WMH and establish a nomogram， and the receiver operating characteristic （ROC） curve and the calibration curve were used to evaluate the diagnostic efficacy of the nomogram. Results Age， course of hypertension， cystatin C， homocysteine，red blood cell distribution width， and cognitive impairment were the independent influencing factors for WMH in the middle-aged and elderly patients with hypertension. The nomogram established showed good diagnostic efficacy （AUC=0.815， 95% CI 0.756~0.874，P<0.001） and calibration ability （C index=0.794）. Conclusion The nomogram established in this study has a good predictive ability for moderate-to-severe WMH in middle-aged and elderly patients with hypertension and can provide certain help for clinical workers.
Nomograms

Objective To investigate the value of ADC histogram in the differential diagnosis of angiomatous meningioma (AM) and hemangiopericytoma (HPC)and to screen out the best diagnostic parameter.Methods The MRI data of 21 cases with AM and 22 cases with HPC confirmed by surgical pathology were analyzed retrospectively.The ROI were drawn on the maximum cross section of the tumor on ADC maps,and the ADC histogram analysis was performed using the software named Mazda.Then the histogram parameters were statistically analyzed to find out the statistically significant parameters between the two groups,and the ROC curve was drawn to evaluate their diagnostic efficacy.Results ADCvariance ,ADC1th and ADC10th had statistical significances between the two groups (P＝0.030,0.002 and 0.02 1 ). ADC1th had the best diagnostic efficacy among them,with the optimal cut-off value of 0.086×10－3 mm2/s,the AUC was 0.814(P＝0.003),and the sensitivity and specificity were 86.70%and 64.70%,respectively.ADCvariance took the second place,for the AUC was 0.725 (P＝0.030),and the sensitivity and specificity were 7 6.50%and 80.00%,respectively.Conclusion The ADC histogram is of great value in the differential diagnosis of AM and HPC,and the ADC1th is the most effective parameter.

Objective To evaluate the effect of remifentanil on iron metabolism in spinal dorsal horn neurons of rats.Methods The primary spinal dorsal horn neurons of rats were seeded in the culture plate at a density of 2× 105 cells/well and divided into 4 groups using a random number table method:control group (C group,n=40),remifentanil group (R group,n=40),divalent metal transporter 1 without iron-responsive element [DMT1 (-) IRE] siRNA group (siRNA group,n=32) and DMT1 (-) IRE siRNA plus remifentanil group (siRNA+R group,n=32).siRNA and siRNA+R groups were subjected to DMT1 (-) IRE siRNA transfection on day 3 of culture.R and siRNA+R groups were incubated for 60 min in the solution with remifentanil at a final concentration of 40 nmol/L.The contents of reactive oxygen species (ROS) and Fe2+ were determined by fluorescent probe method,the malondialdehyde (MDA) content was detected by TBA method,and the content of labile iron pool (LIP) was detected by calcein AM and iron chelator at the end of incubation with remifentanil in R and siRNA+R groups and at the corresponding time points in the other groups.The expression of DMT1 (-) IRE and DMT1 (+) IRE was determined by Westem blot in C and R groups.Results Compared with C group,the expression of DMT1 (-) IRE in the spinal dorsal horn neurons was significantly up-regulated,the contents of Fe2+,LIP,ROS and MDA were increased (P＜0.05),and no significant change was found in the expression of DMT1 (+) IRE in R group (P＞0.05).Compared with R group,the contents of Fe2+,LIP,ROS and MDA in the spinal dorsal horn neurons were significantly decreased in siRNA+R group (P＜0.05).Conclusion Remifentanil increases the iron content of spinal dorsal horn neurons by activating DMT1 (-) IRE,which may be associated with the mechanism of remifentanil-induced postoperative hyperalgesia in rats.

Objective To evaluate the changes in the expression of artemin in skin around the inci-sion during remifentanil-induced hyperalgesia in the rats with incisional pain. Methods Thirty-two healthy male Sprague-Dawley rats, aged 10-12 weeks, weighing 250-280 g, were divided into 4 groups (n = 8 each) using a random number table: control group (group C), incisional pain group (group I), remifen-tanil group (group R) and incisional pain plus remifentanil group (group I+R). Remifentanil was intrave-nously infused for 60 min at a rate of 1 μg·kg-1 ·min-1 in group R. In group I, the model of incisional pain was established, and the equal volume of normal saline was infused for 60 min via the tail vein at the same time. In group I+R, the model of incisional pain was established, and remifentanil was infused for 60 min via the tail vein at a rate of 1 μg·kg-1 ·min-1 at the same time. The equal volume of normal saline was infused for 60 min via the tail vein in group C. Mechanical paw withdrawal threshold (MWT) and ther-mal paw withdrawal latency (TWL) were measured at 24 h before infusion of remifentanil or normal saline and 2, 6, 24 and 48 h after the end of infusion (T0-4 ). Rats were sacrificed following the last measurement of pain threshold, and ipsilateral plantar skin was removed for detection of the expression of artemin protein and mRNA (by fluorescent quantitative real-time polymerase chain reaction or Western blot). Results Compared with group C, MWT was significantly decreased and TWL was shorten at T1-4 , and the expression of artemin protein and mRNA in plantar skin was up-regulated in R, I and I+R groups (P<0. 01). Compared with R and I groups, MWT was significantly decreased and TWL was shorten at T1-4 , and the ex-pression of artemin protein and mRNA in plantar skin was up-regulated in group I+R (P<0. 01). Conclu-sion The peripheral mechanism by which remifentanil induces hyperalgesia may be related to up-regulated expression of artemin in skin around the incision in the rats with incisional pain.

Objective To evaluate the role of spinal C-C motif chemokine receptor 5 (CCRS) in remifentanil-induced hyperalgesia in rats with incisional pain.Methods Thirty-two male Sprague-Dawley rats,weighing 240-260 g,aged 2-3 months,in which intrathecal and caudal catheters were successfully implanted,were divided into 4 groups (n =8 each) using a random number table:control group (group C),CCR5 antagonist maraviroc group (group M),remifentanil plus incisional pain group (group R + I) and maraviroc plus remifentanil plus incisional pain group (group M + R + I).Phosphate buffer solution (PBS) 10 μl was intrathecally injected and normal saline was infused for 60 rnin at 1 μg · kg-1 · min-1 via the caudal vein in group C.Maraviroc 100 pmol (in 10 μl of PBS) was intrathecally injected and normal saline was infused for 60 min at 1 μg · kg-1 · min-1 via the caudal vein in group M.PBS 10 μl was intrathecally injected,then the model of incisional pain was established,and remifentanil 1 μg · kg-1 · min-1 was infused for 60 min via the caudal vein in group R+I.Maraviroc 100 pmol (in 10 μl of PBS) was intrathecally injected,then the model of incisional pain was established,and remifentanil 1 μg · kg-1 · min-1 was infused for 60 min via the caudal vein in group M+R+I.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 24 h before infusion of remifentanil or normal saline (T0) and 2,6,24 and 48 h after stopping infusion (T1-4).The rats were sacrificed after the last measurement of pain threshold,and L4-6 segments of the spinal cord were removed for determination of the expression of glial fibrillary acidic protein (GFAP) and ionized calciumbinding adapter molecule-1 (Iba-1) by Western blot.Results Compared with group C,the MWT was significantly decreased and TWL was shortened at T1-4,and the expression of GFAP and Iba-1 in the spinal cord was up-regulated in R+I and M+R+I groups (P＜0.05),and no significant change was found in the parameters mentioned above in group M (P＞0.05).Compared with group R+I,the MWT was significantly increased and TWL was prolonged at T1-4,and the expression of GFAP and Iba-1 in the spinal cord was down-regulated in group M+R+I (P＜0.05).Conclusion Spinal CCR5 is involved in remifentanil-induced hyperalgesia in the rats with incisional pain,and the mechanism may be related to activating astrocytes and microglias.

Based on WeChat platform,blended teaching is designed and implemented in oral surgery practice.The undergraduate intern can get in touch with the internship early,become familiar with it early and make an internship program early.In the process of internship,they can achieve the purpose of getting more questions,more summary and more test through teaching,discussing and contesting.After the internship,they can consolidate,review,exchange and expand learning.The preliminary practice shows that the teaching stimulates the students' interest and motivation in the clinical study of oral surgery,enhances the interaction between teachers and students and the supervision of the teachers to the students,and improves the effect of the practice teaching.

Objective To construct a technical platform for scarless gene modification of Yersinia pestis and to study the functions of its specific genes.Methods The resistance fragment, including upstream and downstream homologous arms of targeted regions, was reamplified by asymmetric PCR.The amplicons were introduced into Y.pestis harboring plasmid pKD46.With the induction of L-arabinose,the recombinant related enzymes: Exo, Beta and Gam, were expressed to guide the homologous recombination.A donor plasmid, pKSI-1, which carried the desired modification fragment flanking by I-SceⅠ recognition sites, was introduced into Y.pestis as the second step of λ-Red recombination with the help of pREDTKI.Results and Conclusion Two mutant strains:△waaA and waaA(△9nt), were successfully constructed for Y.pestis strain 201.Scarless modification introduces no extra modification to the genome, and it is ideal for comprehensive functional genomic studies.