ObjectiveUsing the Delphi method and analytic hierarchy process （AHP） to screen diagnostic items for qi stagnation syndrome and determine their weights， providing a reference for the development of a diagnostic scale of qi stagnation syndrome. MethodsLiterature related to qi stagnation syndrome were screened from databases including CNKI， Wanfang， VIP， SinoMed （from inception to October 31， 2020）. Through systematic review of literature and expert discussions， the information on the four examinations of traditional Chinese medicine were organized and an item pool was constructed. The Delphi method was used to screen the item indicators， while the AHP was employed to determine their weights. Statistical methods such as mean value， full score ratio， rank sum， unimportant percentage， and coefficient of variation were used for item screening， with the weights calculated by AHP serving as the item weights. ResultsA total of 235 articles and books were included for analysis， resulting in an item pool of 16 items. After three rounds of expert consultation， a total of 84 valid questionnaires were collected， with a total expert enthusiasm coefficient of 99% and authority coefficient of 0.86， 0.84， 0.83， respectively， and the coordination coefficients were 0.45， 0.49， and 0.29， respectively. Through the statistics analysis， 8 diagnosis items were screened out， including distension （stuffi-ness） or distending pain or scurrying pain， wiry pulse， depressed emotions， frequent sighing， deep and wiry pulse， irritability， pale red tongue， and thin white coating. The AHP showed that the order of weights of the first-level indicators from high to low was clinical symptoms， pulse manifestation， and tongue manifestation； the order of weights of the second-level indicators from high to low was distension （stuffiness） or distending pain or scurrying pain， wiry pulse， depressed emotions， frequent sighing， deep and wiry pulse， irritability， pale red tongue， and thin white coating. ConclusionBy applying the Delphi method and AHP to analyze and evaluate the diagnostic items for qi stagnation syndrome， key diagnostic items were screened and their weights determined， laying the foundation for the development of a diagnostic scale for qi stagnation syndrome.

Objective:To explore the effect of significant variation in non-structural protein 1 (NSP1) of 2019 novel coronavirus (2019-nCoV) on binding to 5′UTR, and to provide clues for the development of antiviral drugs and vaccines.Methods:The bioinformatics analysis of 2019-nCoV genome database was conducted to select the amino acid variation sites (T12A, R124L, N128I, K141A, GHVMV82-86DEL and KSF141-143DEL) that may affect the structure of NSP1 and the ability binding to 5′UTR. PSIPRED online tool was used to analyze the secondary structure of the variants, mCSM-NA was used to predict their binding ability to RNA, and DynaMut webserver was used to analyze the influence of variants on protein stability. The variant plasmids were constructed and transfected into HEK-293T cells. The dual luciferase reporter gene assay and RNA binding protein immunoprecipitation (RIP) assay were used to detect the binding ability of the NSP1 variant for viral 5′UTR.Results:Bioinformatics analysis predicted that except for R124L mutation, the other five variants could change the secondary structure of protein, and the mutations of T12A, R124L, N128I and K141A could reduce the binding ability of RNA, while the mutations of T12A, R124L and N128I reduced the stability of protein. The experimental results showed that R124L, N128I, GHVMV82-86DEL and KSF141-143DEL significantly weakened the binding ability of NSP1 to 5′UTR.Conclusions:Some mutations or deletion of NSP1 amino acids could change the secondary structure and significantly weaken the binding ability of NSP1 to the 5′UTR, suggesting that the pathogenicity of the virus may be changed, which could provide a theoretical basis for the development of antiviral drugs and vaccines.

Objective:To establish a rapid, sensitive and specific detection method for important imported viruses based on the recombinase aided amplification (RAA) method and clustered regularly interspaced short palindromic repeats-associated protein 13a (CRISPR-Cas13a) system.Methods:In this study, we selected Japanese encephalitis virus (JEV), Yellow fever virus (YEV), West Nile virus (WNV), Middle East respiratory syndrome coronavirus (MERS-CoV)、Ebola virus (EBOV), Dengue virus (DENV), Rift Valley fever virus (RVFV), Zika virus (ZIKV) as subjects, and designed specific RAA primers and CRISPR RNA(crRNA). The sensitivity and specificity of the method were evaluated. We detected suspected clinical samples of dengue fever and compared with the fluorescent reverse transcriptase-polymerase chain reaction (RT-PCR) technology. Clinical simulation samples of the remaining seven viruses were also detected.Results:The RAA method combined with CRISPR-Cas13a can detect eight pathogens within 40-52 min at 39 ℃. The sensitivity was 1-10 copies/μl. There was no cross-reaction among eight viruses and all clinical samples could be detected by this method.Conclusions:The established RAA combined with CRISPR-Cas13a detection method can sensitively, specifically and quickly detect eight imported infectious disease pathogens.

Objective:To understand the current situation of physicians′ competency in China and statistically analyze its influencing factors, hence providing referential evidences for promoting their competency.Methods:The evaluation scale for Chinese physicians′ competency was developed by referring to sophisticated overseas evaluation frameworks. Based on the electronic platform of the medical alliance, stratified sampling was conducted at 39 hospitals in 13 provinces, from which 2 156 physicians were surveyed, and statistical analysis was made on the current situation and influential factor of their competency in 6 dimensions(medical knowledge, diagnosis and treatment, scientific research and teaching, interpersonal communication, spirit of professionalism, population health)Results:The score range in multiple dimensions of physicians′ competency was between 2.85 and 3.89(5 in total), among which spirit of professionalism dimension scores the highest, and teaching and research dimension scores the lowest. Logistic regression analysis shows that their competency level in 6 dimensions is correlated to a range of factors, including gender, degree, title, supervisor qualifications, standardized training program of specialist physicians, and affiliation or not to teaching hospitals( P<0.05). Conclusions:Their overall competency is expected to be elevated. To name a few, they are lack of teaching and research abilities, and need to conduct clinical-oriented scientific research; they are expected to enrich knowledge structure, and need to embrace a wider concept of health; they need to improve their non-technical service abilities, and should further emphasize their humanistic quality; physicians′ practice competency depends upon their extent of medical education, and ensuring the quality of standardized training becomes the key measure.

Objective:To establish a rapid method for detecting the activity of 2019-nCoV2.Methods:The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) detection system was screened after propidium monoazide (PMA) treatment and exposure to heat-inactivated 2019-nCoV samples, and the PMA pretreatment conditions were optimized to establish the 2019-nCoV PMA-RT-qPCR detection method . The established method was used to detect virus inactivated by different temperatures and chlorine-containing disinfectant, to evaluate its effect in detecting virus activity.Results:For the PMA-RT-qPCR assay, 200 μmol/L of PMA, 10 min of incubation time, 15 min of exposure time, and the CDC ORF1ab detection system were selected; there was no significant difference in the result of PMA-RT-qPCR and direct RT-qPCR for the active virus; the Ct values of PMA-RT-qPCR for virus inactivated by 95 ℃ and chlorine-containing disinfectant were higher than that of control groups at different dilutions; only partial dilutions of 70 ℃ and 56 ℃ heat-inactivated virus had higher Ct values than control groups. Conclusions:The established PMA-RT-qPCR for 2019-nCoV activity detection method has a good detection effect on the virus inactivated by 95 ℃ heat and chlorine disinfectant, and provides an auxiliary means for judging the infectivity of the virus in the sample.

Objective:To investigate whether lncRNA LINC00909 affected the radiosensitivity of colorectal cancer cells by targeting miR-548-3p.Methods:The expression levels of LINC00909 and miR-584-3p in colorectal cancer and adjacent tissues were detected by qRT-PCR. The colorectal cancer cells SW480 and SW620 were cultured in vitro, and transfected with si-NC, si-LINC00909, miR-NC, miR-584-3p mimics, si-LINC00909, and anti-miR-NC and si-LINC00909, and anti-miR-584-3p, respectively. The cells were irradiated with a dose of 4 Gy. The cell survival fraction and sensitization enhancement ratio (SER) were detected by clone formation assay. Cell proliferation was detected by MTT assay. Cell migration and invasion were assessed by Trans well chamber assay. The targeting relationship between LINC00909 and miR-584-3p was confirmed by dual luciferase reporter assay. The effect of interfering with the expression of LINC00909 or inhibiting the expression of miR-584-3p on the weight of the xenograft tumor after irradiation was evaluated by subcutaneous xenograft experiment in nude mice. Results:The expression level of LINC00909 in colorectal cancer tissues was significantly up-regulated ( P<0.05), whereas the expression level of miR-584-3p was significantly down-regulated ( P<0.05). After interfering with the expression of LINC00909 or miR-584-3p overexpression, the cell survival fraction score was significantly reduced ( P<0.05), the SERs were 2.017 and 1.762, and cell proliferation, migration and invasion were suppressed (all P<0.05). Dual luciferase reporter assay confirmed that LINC00909 could target and bind to miR-584-3p. After interfering with the expression of LINC00909, the weight of the transplanted tumor was significantly reduced ( P<0.05), whereas the weight of the transplanted tumor was significantly increased after co-transfection with anti-miR-584-3p ( P<0.05). Conclusion:Interfering with the expression of LINC00909 can inhibit the proliferation, migration and invasion ability of colorectal cancer cells by up-regulating the expression of miR-548-3p, thereby enhancing the cell radiosensitivity.

Objective:The entire genome sequences of 25 novel coronaviruses in Jiangsu province were analyzed and their evolutionary characteristics were studied.Methods:High-throughput sequencing was used to sequence the throat swab samples from confirmed cases. Single nucleotide polymorphisms were analyzed using CLC Genomics Workbench 12.0 software. Evolution characteristics were analyzed by MEGA 5.1.Results:A total of 52 single-base substitution mutations were detected in 25 strains. Phylogenetic analysis showed 25 stains were clustered into two clades. Viruses in clade 1 contain 8 682 and 28 144 CT SNP links. While viruses in clade 2 contain mutations in those two bases, i. e., 8 682 (ORF1ab: C8 517T, synonymous mutation) and 28 144 (ORF8: T251C, L84S). Among clade 2, five stains subclustered into one group based on SNP links in 24 034 (S: C2 472T, synonymous mutation), 26 729 (M: T207C, synonymous mutation), and 28 077 (ORF8: G184C, V62 L). There were no significant differences in the distribution of different clades/subclusters in the population and the disease types.Conclusions:We have found some SNPs occurred in new coronaviruses. The effects of different SNPs on virus transmission and pathogenicity need to be further studied.

Objective To investigate the effect of lncRNA CCAT1 and miR-130b-3p on the radiosensitivity of human pancreatic cancer cells PANC-1.Methods Real-time PCR was used to detect the relative expression levels of CCAT1 and miR-130b-3p in pancreatic cancer tissues and cell lines including PANC-1 cells irradiated with 2 Gy X-rays.After silencing CCAT1 and/or inhibiting miR-130b-3p expression,cell apoptosis rate,Caspase 3 activity and cell survival were detected by flow cytometry,Caspase 3 activity detection kit and colony formation assay,respectively.Cell survival curve was stimulated by the multi-target single-hit model.Based on the starBase v2.0 online analysis,the luciferase reporter gene assay,RNA-binding protein immunoprecipitation assay (RIP) and Real-time PCR assay were applied to verify the relationship between CCAT1 and miR-130b-3p.Results CCAT1 expression was up-regulated (t=6.322-8.555,P＜0.05),but miR-130b-3p expression was down-regulated (t =3.950-18.795,P＜ 0.05) in the radiation-resistant pancreatic cancer tissues,pancreatic cancer cell lines and 2 Gy-irradiated PANC-1 cells.When the CCAT1 silenced PANC-1 cells were irradiated with 2 Gy,cell survival fraction decreased (t=2.929,5.047,5.234,5.125,P＜0.05),apoptosis rate and Caspase 3 activity increased (t=6.953,6.836,P＜0.05).CCAT1 could selectively regulate miR-130b-3p expression.Inhibition of miR-130b-3p expression could enhance PANC-l cell survival (t =4.564,6.736,8.656,P＜0.05),but reduced apoptosis rate (t=5.234,P＜0.05) and Caspase 3 activity (t=10.440,P＜0.05).Conclusions Silencing CCAT1 promotes the expression of miR-130b-3p and enhances radiosensitivity of PANC-1 cells.

Objective: To investigate the pyroptosis induced by different enteroviruses in human neuroblastoma cells SH-SY5Y and the differences among them. Methods: SH-SY5Y cells were infected with nine strains of enterovirus respectively, including enterovirus A71 (EV-A71), Coxsackievirus A (CA), Coxsackievirus B (CB), Echovirus (Echo). The cellular morphology of infected and control groups were observed and activity of Caspase-1 of infected and control groups were detected by flow cytometry at 48 h post infection. Results: The activity of Caspase-1 induced by EV-A71 was higher than control (P<0.001), and the activity of Caspase-1 induced by EV-A71 isolated from severe case was significantly higher than that induced by EV-A71 isolated from common case (P<0.001). The activity of Caspase-1 induced by CA was at a lower level. The activity of Caspase-1 induced by CB and Echo were both significantly higher than that induced by EV-A71 and control (P<0.001). Cytopathic effects (CPE) were found to be related with the activity of Caspase-1. Conclusions EV-A71, CB and Echo all could induce pyroptosis mediated by Caspase-1 in SH-SY5Y cells, but the ability of CA to induce pyroptosis was at a lower level, which may provide evidences for further study on mechanism of neuropathy caused by enterovirus.

Objective: To investigate the effect and potential mechanism of HOXC8 on the radiosensitivity of non-small cell lung cancer cell line A549, aiming to provide novel ideas for clinical combined treatment. Methods: The A549 cells with stable knockdown of HOXC8 were constructed by using lentivirus and validated by qPCR and Western blot. The radiosensitivity of A549 stable cell line was assessed by plate clone formation assay. The expression levels of TGF-β₁ and the proteins in the downstream signal pathway after knockdown of HOXC8 were detected by Western blot. Results: The A549 cells with stable knockdown of HOXC8 were successfully constructed. The viability and clonogenic capacity of A549 cells were significantly reduced after silencing HOXC8. Silencing HOXC8 also increased the sensitivity of A549 cells to radiotherapy and significantly inhibited the expression of TGF-β₁ and p-Smad2/3 proteins in the downstream signaling pathway. Conclusion Silencing HOXC8 can increase the sensitivity of A549 cells to radiotherapy probably by inhibiting TGF-β₁ signaling transduction. HOXC8 might play an important role in A549 cells.