Objective:To investigate the dynamic changes of cellular miRNA expression profiles in EBV latently infected Raji cells upon reactivation with Phorbol ester (TPA).Methods:Total RNA was extracted using TRIzol reagent from Raji cells treated with TPA at different time points (0 h, 24 h, 48 h). Small RNA libraries were constructed and sequenced on an Illumina SE50 platform. Differentially expressed miRNAs were identified, and their target genes were predicted and functionally annotated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were carried out through online tools. Additionally, miRanda and RNAhybrid software were used to predict cellular miRNAs targeting the EBV genome. Real-time RT-qPCR was employed to validate the expression levels of differentially expressed novel miRNAs.Results:High-throughput sequencing identified 1 301 celluar miRNAs, comprising 1 189 known and 112 novel miRNAs. A total of 264 known differentially expressed cellular miRNAs and 13 novel miRNAs were identified through high-throughput miRNA sequencing. Secondary structure prediction revealed that the novel miRNAs exhibited typical pre-miRNA hairpin structures. Stem-loop quantitative real-time PCR (RT-qPCR) validation of Novel_miR_183 and Novel_miR_242 did not exhibit a statistically significant difference ( F=1.407, P=0.370 7 for Novel_miR_183; F=1.277, P=0.397 0 for Novel_miR_242) between the TPA-stimulated and untreated groups. Target gene prediction analysis revealed that the differentially expressed cellular miRNAs were involved in various important biological processes and signaling pathways. Furthermore, 1 189 known cellular miRNAs and 108 novel miRNAs were predicted to target the EBV genome. Conclusions:Treatment of Raji cells with TPA stimulation successfully reactivated Raji cells and significantly altered their miRNA expression patterns. Differentially expressed miRNAs were identified, suggesting that these miRNAs probably play crucial roles in regulating EBV infection and replication by directly targeting the EBV genome.

Objective:To investigate the effect of nuclear factor erythroid-2 related factor (Nrf2) overexpression on high-risk human papillomavirus type 16 (HPV16) E6 protein.Methods:The pRK5-FLAG-NFE2L2 plasmid was constructed, and pRK5-FLAG-NFE2L2 and pRK5-HA-HPV16E6 plasmids were co-transfected in HEK293T cells, and the expression of the two proteins was detected by Western blotting (WB). Next, pRK5-HA-HPV16E6 and pRK5-FLAG-Nrf2 plasmids were expressed using an in vitro transcription system to observe the expression of both. Finally, pEGFP-HPV16E6 and pLVX-mCherry-Nrf2 plasmids were co-transfected in HEK293T cells, and the cellular localization of the E6 protein and the Nrf2 protein was observed using fluorescence microscopy.Results:Nrf2 protein was successfully overexpressed in HEK293T cells, and the WB result showed that Nrf2 inhibited HPV16 E6 protein expression in a significant dose-dependent manner. The expression of HPV16 E6 protein in the TNT Quick Coupled Transcription/Translation Systems was affected by Nrf2, while the expression of HPV16 E6 in TnT SP6 High-Yield Wheat Germ Protein Expression System was no longer inhibited by Nrf2. Fluorescence imaging further showed no intracellular co-localization of Nrf2 and HPV16 E6.Conclusions:Overexpression of the nuclear transcription factor Nrf2 reduces HPV16 E6 protein expression, but there is no intracellular co-localization of them.

Objective:To establish a protocol for virus isolation using the mixed method, and evaluate the efficacy of the suspended method and the mixed method in isolating herpes simplex virus (HSV).Methods:Simulated HSV-infected clinical samples were prepared using HSV-1 F strain and CDC-P1 strain. Both the suspended method and the mixed method were used to isolate HSV-1 from these samples. The virus isolation efficiency of the mixed method under various conditions was assessed. These conditions included different multiplicity of infection (MOI), cell seeding densities, and virus adsorption times.The 50% tissue culture infective dose (TCID 50) assay was used for the assessment. The positive rates of virus detection under low viral load conditions were compared between the two methods. Results:Under the conditions of a MOI of 0.005, a virus adsorption time of 15 min, and a cell seeding density of 1×10 6 cell/ml, the mixed method achieved effective isolation of HSV-1. When the virus titer of the sample was 100 TCID 50/ml, the positivity rate of the mixed method reached 100.0%, while the positivity rates of the suspended method were 50.7% (38/75) and 52.0% (39/75) after cultured for 72 h and 96 h, respectively. When the virus titer of the sample was 10 TCID 50/ml, the positivity rate of the mixed method was 100.0%, while the positivity rate of the suspension method was 0. Conclusions:The mixed method exhibits significantly higher efficiency in HSV isolation compared with the suspended method. Under the conditions of high viral load, both the suspended method and the mixed method can be effective in isolating HSV-1. For clinical samples with low viral loads, the mixed method has greater applicability.

OBJECTIVE To investigate the changes of air aerosols in the digestive endoscope cleaning and disinfec-tion room under real working conditions and observe the purification effects of aerosols under different ventilation and disinfection modes.METHODS Under the real working conditions(with both the air conditioner and recircu-lating air disinfection machine under the working mode),the air samples were collected every 1 hour from 8:00 to 16:00 from the digestive endoscope cleaning and disinfection room of Ren Ji Hospital,Shanghai Jiao Tong U-niversity School of Medicine from May 2023 to Oct.2023;the contents of particulate matters(PM)and microor-ganisms in the air were detected.At the busiest moment of the cleaning and disinfection room,the air was respec-tively sampled from the cleaning and disinfection room with the working condition modes of air condition systems,recirculating air disinfection machine or natural ventilation before the starting of systems and after the work for 0.5 hour,1 hour and 2 hours.The contents of PMs and microorganisms were detected.RESULTS The PM0.5 con-centrations during various time periods met the Grade 9 ISO clean room standard(≤35,200 particles/L)under the real working conditions,and the content of airborne viable particles also conformed to the Class Ⅲ environmental requirements(≤500 CFU/m3).All of the three ventilation and disinfection methods showed certain effects on purification of the con-tents of PM0.5 and microorganisms after the ventilation for 2 hours,the contents of PMs and microorganisms of the air disinfection machine group decreased most remarkably,followed by the air conditioner group,the natural ventilation group the least.There were no significant differences in the PM concentration and the content of microorganisms among the three groups at the time points;there were only significant differences in the PM concentration and the content of mi-croorganisms of the air disinfection machine group after the ventilation for 2 hours,0.5 hour and 1 hour(P＜0.05).CONCLUSIONS Both the air conditioning systems and recirculating air disinfection machine under the working con-dition mode have certain effect on purification of the contents of PM and microorganisms,and the specific effect need to be further studied.The implementation of multicenter dynamic surveillance with the help of intelli-gent techniques may provide reference for the optimization of ventilation and disinfection strategies.

OBJECTIVE To investigate the changes of air aerosols in the digestive endoscope cleaning and disinfec-tion room under real working conditions and observe the purification effects of aerosols under different ventilation and disinfection modes.METHODS Under the real working conditions(with both the air conditioner and recircu-lating air disinfection machine under the working mode),the air samples were collected every 1 hour from 8:00 to 16:00 from the digestive endoscope cleaning and disinfection room of Ren Ji Hospital,Shanghai Jiao Tong U-niversity School of Medicine from May 2023 to Oct.2023;the contents of particulate matters(PM)and microor-ganisms in the air were detected.At the busiest moment of the cleaning and disinfection room,the air was respec-tively sampled from the cleaning and disinfection room with the working condition modes of air condition systems,recirculating air disinfection machine or natural ventilation before the starting of systems and after the work for 0.5 hour,1 hour and 2 hours.The contents of PMs and microorganisms were detected.RESULTS The PM0.5 con-centrations during various time periods met the Grade 9 ISO clean room standard(≤35,200 particles/L)under the real working conditions,and the content of airborne viable particles also conformed to the Class Ⅲ environmental requirements(≤500 CFU/m3).All of the three ventilation and disinfection methods showed certain effects on purification of the con-tents of PM0.5 and microorganisms after the ventilation for 2 hours,the contents of PMs and microorganisms of the air disinfection machine group decreased most remarkably,followed by the air conditioner group,the natural ventilation group the least.There were no significant differences in the PM concentration and the content of microorganisms among the three groups at the time points;there were only significant differences in the PM concentration and the content of mi-croorganisms of the air disinfection machine group after the ventilation for 2 hours,0.5 hour and 1 hour(P＜0.05).CONCLUSIONS Both the air conditioning systems and recirculating air disinfection machine under the working con-dition mode have certain effect on purification of the contents of PM and microorganisms,and the specific effect need to be further studied.The implementation of multicenter dynamic surveillance with the help of intelli-gent techniques may provide reference for the optimization of ventilation and disinfection strategies.

Objective:To establish a protocol for virus isolation using the mixed method, and evaluate the efficacy of the suspended method and the mixed method in isolating herpes simplex virus (HSV).Methods:Simulated HSV-infected clinical samples were prepared using HSV-1 F strain and CDC-P1 strain. Both the suspended method and the mixed method were used to isolate HSV-1 from these samples. The virus isolation efficiency of the mixed method under various conditions was assessed. These conditions included different multiplicity of infection (MOI), cell seeding densities, and virus adsorption times.The 50% tissue culture infective dose (TCID 50) assay was used for the assessment. The positive rates of virus detection under low viral load conditions were compared between the two methods. Results:Under the conditions of a MOI of 0.005, a virus adsorption time of 15 min, and a cell seeding density of 1×10 6 cell/ml, the mixed method achieved effective isolation of HSV-1. When the virus titer of the sample was 100 TCID 50/ml, the positivity rate of the mixed method reached 100.0%, while the positivity rates of the suspended method were 50.7% (38/75) and 52.0% (39/75) after cultured for 72 h and 96 h, respectively. When the virus titer of the sample was 10 TCID 50/ml, the positivity rate of the mixed method was 100.0%, while the positivity rate of the suspension method was 0. Conclusions:The mixed method exhibits significantly higher efficiency in HSV isolation compared with the suspended method. Under the conditions of high viral load, both the suspended method and the mixed method can be effective in isolating HSV-1. For clinical samples with low viral loads, the mixed method has greater applicability.

Objective:To investigate the dynamic changes of cellular miRNA expression profiles in EBV latently infected Raji cells upon reactivation with Phorbol ester (TPA).Methods:Total RNA was extracted using TRIzol reagent from Raji cells treated with TPA at different time points (0 h, 24 h, 48 h). Small RNA libraries were constructed and sequenced on an Illumina SE50 platform. Differentially expressed miRNAs were identified, and their target genes were predicted and functionally annotated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were carried out through online tools. Additionally, miRanda and RNAhybrid software were used to predict cellular miRNAs targeting the EBV genome. Real-time RT-qPCR was employed to validate the expression levels of differentially expressed novel miRNAs.Results:High-throughput sequencing identified 1 301 celluar miRNAs, comprising 1 189 known and 112 novel miRNAs. A total of 264 known differentially expressed cellular miRNAs and 13 novel miRNAs were identified through high-throughput miRNA sequencing. Secondary structure prediction revealed that the novel miRNAs exhibited typical pre-miRNA hairpin structures. Stem-loop quantitative real-time PCR (RT-qPCR) validation of Novel_miR_183 and Novel_miR_242 did not exhibit a statistically significant difference ( F=1.407, P=0.370 7 for Novel_miR_183; F=1.277, P=0.397 0 for Novel_miR_242) between the TPA-stimulated and untreated groups. Target gene prediction analysis revealed that the differentially expressed cellular miRNAs were involved in various important biological processes and signaling pathways. Furthermore, 1 189 known cellular miRNAs and 108 novel miRNAs were predicted to target the EBV genome. Conclusions:Treatment of Raji cells with TPA stimulation successfully reactivated Raji cells and significantly altered their miRNA expression patterns. Differentially expressed miRNAs were identified, suggesting that these miRNAs probably play crucial roles in regulating EBV infection and replication by directly targeting the EBV genome.

Objective:To investigate the effect of nuclear factor erythroid-2 related factor (Nrf2) overexpression on high-risk human papillomavirus type 16 (HPV16) E6 protein.Methods:The pRK5-FLAG-NFE2L2 plasmid was constructed, and pRK5-FLAG-NFE2L2 and pRK5-HA-HPV16E6 plasmids were co-transfected in HEK293T cells, and the expression of the two proteins was detected by Western blotting (WB). Next, pRK5-HA-HPV16E6 and pRK5-FLAG-Nrf2 plasmids were expressed using an in vitro transcription system to observe the expression of both. Finally, pEGFP-HPV16E6 and pLVX-mCherry-Nrf2 plasmids were co-transfected in HEK293T cells, and the cellular localization of the E6 protein and the Nrf2 protein was observed using fluorescence microscopy.Results:Nrf2 protein was successfully overexpressed in HEK293T cells, and the WB result showed that Nrf2 inhibited HPV16 E6 protein expression in a significant dose-dependent manner. The expression of HPV16 E6 protein in the TNT Quick Coupled Transcription/Translation Systems was affected by Nrf2, while the expression of HPV16 E6 in TnT SP6 High-Yield Wheat Germ Protein Expression System was no longer inhibited by Nrf2. Fluorescence imaging further showed no intracellular co-localization of Nrf2 and HPV16 E6.Conclusions:Overexpression of the nuclear transcription factor Nrf2 reduces HPV16 E6 protein expression, but there is no intracellular co-localization of them.

Objective: To investigate the clinical values of colposcopy and cervical biopsy and/or endocervical curettage (ECC) in the diagnosis of cervical lesion. Methods: Clinical data of 128 cases of cervical lesion diagnosed by Xuzhou Cancer Hospital from January 23, 2014 to October 11, 2016 were collected and retrospectively analyzed, all patients underwent colposcopy and cervical biopsy and/or ECC. Results: Among them, the age between 30 to 50 years old were 70 cases, whose transformation zone types of Ⅰ, Ⅱ and Ⅲ were 28 cases (40.0%), 23 cases (32.9%) and 19 cases (27.1%), respectively. The age older than 50 years were 45 cases, whose transformation zone types of Ⅱ and Ⅲ were 1 case (2.2%) and 44 cases (97.8%), respectively. Among the 128 cases of cervical lesions, diagnostic results of colposcopy showed that the chronic inflammation were 57 cases, cervical intraepithelial neoplasia (CIN)Ⅰwere 35 cases, CINⅡor CINⅡ~Ⅲ were 8 cases, CIN Ⅲ were 5 cases and cervical cancer were 23 cases. Alternatively, the pathological results showed that the chronic inflammation were 81 cases, CINⅠwere 17 cases, CINⅡor CINⅡ~Ⅲ were 7 cases, CIN Ⅲ were 5 cases and cervical cancer were 18 cases, respectively. Among the 81 cases of chronic inflammation diagnosed by pathology, 52 cases (64.2%) were consistent with the diagnostic results of colposcopy. Among the 17 cases of low grade squamous epithelial cell lesion (LSIL) diagnosed by pathology, 10 cases were in agree with the diagnostic results of colposcopy. Among the 12 cases of high-grade squamous epithelial cell lesion (HSIL) diagnosed by pathology, 9 cases were concordant with the diagnostic results of colposcopy. Among the 18 cases of cervical cancer diagnosed by pathology, 17 cases were consistent with the diagnostic results of colposcopy. Conclusions The type of transformation zone is positively correlated with the age, and it can help to choose biopsy and therapeutic manner. The diagnostic accuracies of HSIL and early stage of cervical cancer by multi-point biopsy of colposcopy and/or ECC are high. The cervical lesions which are difficultly found by direct visualization can be identified by colposcopy, and thus provides objective evidence to determine the therapeutic manner for patients with stage ⅡA of cervical cancer.

In view of the situation such as the serious shortage of anatomical teachers in medical colleges and universities,irrational personnel structure,anatomical teachers' single knowledge and weak scientific research ability,etc.,we analyzed the national policy,social impact,school leadership,personal career planning and other aspects of the problem and put forward some countermeasures to improve the treatment,improve the environment and train talents,which provided reference for the development of the discipline of anatomy,the construction of teaching staff and the reform of the basic medical education.