Pseudorabies virus(PRV)is the etiological agent of pseudorabies in pigs,which is char-acterized by dyspnea,reproductive disorders,and neurological diseases,and it spreads widely a-round the world.Since 2011,the newly emerged PRV variants have resulted in poor immunity pro-tection of traditional vaccine strains,and the original method of vaccine strain preparation is time-consuming and labor-intensive.Therefore,it is urgently needed to develop an efficient screening method of the vaccine strain at present.Using CRISPR/Cas9 gene editing technology in this study,two single guide RNAs(sgRNA)were designed targeting the virulence gene TK of PRV variant strain CH/JX/2016,and then the enhanced green fluorescent protein the reporter(EGFP)gene was inserted at the TK locus by a homologous repair plasmid.After multiple rounds of plaque puri-fication,the rPRV-ΔTK/EGFP strain was obtained.The results showed the cleavage efficiency of the two sgRNAs was extremely high.The preparation of rPRV-ΔTK/EGFP strain was succeed af-ter only three rounds of purification,and the EGFP expressed normally.The CRISPR/Cas9 system can edit the PRV gene simply,rapidly,and efficiently,and exhibits great potential in the construction of vaccine candidate strains.Meanwhile,the rescued rPRV-ΔTK/EGFP strain not only could be used as a tracer strain in PRV variant infection progresses,but also for subsequent antivi-ral drug screening.

Objective:To establish a polymerase chain reaction(PCR)method to accurately discriminate the crude materials and aqueous extract of Zaocys dhumnades,Elaphe carinata,Elaphe meollendorff and Ptyas korros.Methods:Specific primers were designed using mitochondrial Cytb gene(CO1)as a target gene,and annealing temperature,number of cycles and the type of DNA polymerases were optimized.The mixed samples were detected by this method.Results:By this multiplex allele-specific PCR identification method,135,182,246 and 197 bp of specific fragments were amplified from DNA templates of Zaocys dhumnades,Elaphe carinata,Elaphe meollendorffi and Ptyas korros,respectively,following the conditions:cycle number of 35,annealing temperature of 62 ℃.The adulterants and the blank control showed no bands.The method could simultaneously and accurately identify the snake-derived components in the mixed samples.Conclusion:The method can be used to identify the samples of Zaocys dhumnades,Elaphe carinata,Elaphe meollendorffi and Ptyas korros simultaneously,accurately and rapidly,and is suitable for the identification of standard decoctiond and formula granules samples.

Objective:To verify the prognostic significance of the tumor regression grade (TRG) for muscle-invasive bladder cancer (MIBC) patients undergoing radical cystectomy (RC) after neoadjuvant chemotherapy.Methods:The data of 70 MIBC patients treated with gemcitabine combined with cisplatin neoadjuvant chemotherapy and RC in Sun Yat-sen University Cancer Center between July 2016 to November 2021 were retrospectively reviewed. There were 65 males and 5 females, with an average age(59.79±10.56)years old. The patients accepted transurethral resection of bladder tumor (TURBT) specimens before neoadjuvant chemotherapy. Clinicopathological characteristics of patients were recorded and TRG was assessed. TRG evaluation criteria: TRG 1 was defined as no cancer residue, TRG 2 was defined as the proportion of residual cancer area to tumor bed area <50%, and TRG 3 was defined as the proportion of residual cancer area to the area of the tumor bed ≥ 50%. Chi-square test or Fisher's exact test were used to compare the relationship between patients' clinicopathological characteristics and TRG. The relationship between post-neoadjuvant therapy tumor and node(ypTN)stage, and survival, including overall survival(OS)and recurrence-free survival (RFS) were analyzed by Kaplan-Meier analysis. The pathologically locally descending disease was defined as (ypT < T 2 and ypN=N 0) and pathologically locally advanced disease was defined as (ypT≥T 2 and/or ypN ≥N 1). Cox regression was used for univariate and multivariate analysis of OS and RFS. Results:Chi-square test or Fisher exact test analysis showed TRG was significantly associated with ypT stage ( P < 0.001), ypN stage ( P = 0.002), lympho-vascular invasion ( P<0.001) and variant histology ( P<0.001). The OS of patients with TRG 1, TRG 2 and TRG 3 were 20.5(10.3, 31.8), 17.0(11.0, 30.8)and 15.0(11.0, 26.0) months, respectively, and the difference was significantly different( P = 0.037). The RFS of patients with TRG 1, TRG 2 and TRG 3 were 15.0(8.3, 25.5), 15.0(8.0, 27.0)and 11.0(4.5, 25.5) months, respectively, and the difference was significantly different ( P=0.029). There were significant differences between patients with pathologically locally descending disease and locally advanced disease in OS [18.5(10.3, 30.8)vs.15.0(11.0, 27.3)months, P = 0.013] and RFS [14.0(8.0, 24.0)vs. 11.5(8.0, 26.8)months, P = 0.012]. Among patients with locally advanced pathology, the OS was 19.5(11.0, 32.5)months for patients with TRG ≤2, 13.5(10.8, 26.0)months for patients with TRG 3( P=0.140). The RFS was 12.0(8.0, 31.0)months for those patients with TRG ≤2 and 11.0(6.0, 26.0)months for those patients with TRG 3( P = 0.180). Cox univariate analyses showed that patients with TRG 3 were associated with decreased OS ( HR = 6.043, 95% CI 1.170-31.213, P = 0.032) and RFS ( HR = 6.354, 95% CI 1.231-31.802, P = 0.027). Conclusions:This study showed that TRG was correlated with OS and RFS among patients. The patients who had the higher TRG had the worse prognosis. It was confirmed that TRG predicted the prognosis of patients undergoing radical cystectomy after neoadjuvant chemotherapy. Therefore, TRG assessment is recommend in pathology report for patients who had radical cystectomy after neoadjuvant chemotherapy.

To investigate the effect of nucleoside diphosphate kinase (NDK) on the synthesis of hyaluronic acid, nucleoside diphosphate kinase gene (ndk) was overexpressed along with the hyaluronic acid-producing genes in recombinant B.subtilis. Two engineered strains named Hp8tg and Pn8tg were constructed.Uniform hyaluronic acid (HA) could be obtained from both engineered strains.HA produced by both recombinant strains was confirmed by monosaccharide composition analysis, Fourier transform infrared spectometry and nuclear magnetic resonance spectroscopy.Inducing conditions of HA fermentation were optimized by response surface methodology.Overexpression of ndk could increase the production and molecular weight of HA by 1.3-fold and 1.1-fold, respectively. This study revealed for the first time that overexpression of ndk could relieve the inhibition effect of uridine diphosphate (UDP) on Class II HA synthase and increase the production and molecular weight of HA, which proves to be an efficient strategy for the production of HA, and the preparation of other polysaccharides.

Objective To investigate the status of maternal iodine deficiency in Lishui City of Zhejiang Province， and explore the effect of maternal iodine deficiency on the growth and development of infants. Methods A total of 209 pregnant women living in Liandu District of Lishui City from January 2017 to December 2018 were selected by stratified sampling method. Urine iodine level in pregnant women and iodine content in edible salt at home were determined to assess the status of iodine deficiency and to analyze the influencing factors. Their babies were followed up for 1 year， and metabolic disease screening and physical examination were carried out to evaluate the growth and development. Results The median of urinary iodine levels in 2017 and 2018 were 165 µg·L^-1 and 192 µg·L^-1， respectively. Both of the values met the standard of iodine sufficiency. The results of salt iodine content in pregnant women's homes showed that 7 cases （3.35%） were without iodized salt and 202 cases （96.65%） with iodized salt， including 186 cases （89.00%） with qualified iodized salt. Univariate analysis showed that pregnancy， education level， intake of iodized salt， cooking methods of salt， intake frequency of iodine-rich food and knowledge of iodine nutrition had significant impact on iodine deficiency in pregnant women （P<0.05）. Logistic regression analysis showed that early pregnancy （OR=1.626） and adding salt into heat oil （OR=1.874） were risk factors for iodine deficiency in pregnant women， while intake of qualified iodized salt （OR=0.593） was a protective factor. Follow-up results showed that there were no abnormalities in newborn screening， and babies of 2 women （2.41%） with iodine deficiency were short in height at 1， 6 and 12 months of age. Conclusion Attention should be paid to the monitoring of pregnant women's urinary iodine levels to guide the scientific supplementation of iodine， so as not to affect the growth and development of infants.

Objective To analyze the basic characteristics, drug features, prescription rules, and drug-symptom relationships of patients in the splenic deficiency and impairment stage, by data mining of medical records under the New Theory on Spleen Dampness Syndrome (Pi Dan Xin Lun, 《脾瘅新论》). Methods Medical records listed in the “New Theory on Spleen Dampness Syndrome – Understanding and Treatment of Metabolic Syndrome from the Perspective of Traditional Chinese Medicine”, and which were diagnosed with the spleen dampness syndrome at the splenic deficiency and impairment stage, during January 2004 and December 2016 were selected. These patients’ data, including basic information, clinical symptoms, laboratory examination results, traditional Chinese medicine (TCM) and western medicine diagnoses, treatment methods, prescriptions, etc., were collected. The collected data were subsequently compiled into a medical record database using the Epidata 3.1 data management software, followed by the use of Apriori algorithm provided in the SPSS Modeler 14.2 statistical software to investigate the association rules between drug-drug, drug-symptom, and drug-western medicine indices. Results (i) A total of 51 medical records were included, involving 17 types of syndromes. Among them, the top three with frequency ≥ 3 included “Phlegm and blood stasis, and thoracic obstruction” “Deficiency-weakness of the spleen Qi, and static blood blocking collaterals”, and “Deficiency-weakness of the spleen Qi, and static blood blocking collaterals”. Alternatively, of the 14 treatment methods, the top three treatments with frequency of ≥ 3 included “Activating Yang and eliminating turbidity, and removing phlegm and dredging channel blockage” “Strengthening the spleen and benefiting Qi, and eliminating phlegm to activate the channels”, and “Warming Yang and benefiting Qi, and expelling cold to remove obstructions”. Among the 15 prescriptions, the top three used with frequency ≥ 3 included Huangqi Guizhi Wuwu Tang (黄芪桂枝五物汤), Gualou Xiebai Banxia Tang (瓜蒌薤白半夏汤), and Ganjiang Huangqin Huanglian Renshen Tang (干姜黄芩黄连人参汤). Lastly, of the 83 drugs used for a total of 476 times, those with frequency ≥ 15 included Huanglian (Coptidis Rhizoma), Huangqi (Astragali Radix), Jiudahuang (Wine-processed Rhei Radix et Rhizoma), Jixueteng (Spatholobi Caulis), Shengjiang (Zingiberis Rhizoma Recens), Huangqin (Scutellariae Radix), and Guizhi (Cinnamomi Ramulus). (ii) For the drug-drug associations, under the criteria of support ≥ 15% and confidence = 100%, seven second-order association rules, seven third-order rules, and six fourth-order roles were identified. The top-ranking rule of each was “Huangqin (Scutellariae Radix) → Huanglian (Coptidis Rhizoma)” “Ganjiang (Zingiberis Rhizoma) + Huangqin (Scutellariae Radix) → Huanglian (Coptidis Rhizoma)”, and “Baishao (Paeoniae Radix Alba) + Guizhi (Cinnamomi Ramulus) + Jixueteng (Spatholobi Caulis) → Huangqin (Scutellariae Radix)”, respectively. Alternatively, the drug-symptom associations were analyzed under the criteria of support ≥ 5% and confidence = 100%, which derived eight second-order association rules, 31 third-order rules, and 30 fourth-order rules. The top-ranking association rule of each order was “Huangqi (Astragali Radix) → Limb edema” “Guizhi (Cinnamomi Ramulus) + Jixueteng (Spatholobi Caulis) → Limb numbness and pain”, and “Guizhi (Cinnamomi Ramulus) + Jixueteng (Spatholobi Caulis) + Huangqi (Astragali Radix) → Limb numbness and pain”, respectively. Similarly, the drug-western medicine index associations were investigated under the criteria of support ≥ 5% and confidence = 100%, and five second-order association rules, 16 third-order rules, and 16 fourth-order rules were identified. In this category, the top-ranking association rule of each order was “Qinpi (Fraxini Cortex) → Uric acid” “Huanglian (Coptidis Rhizoma) + Ganjiang (Zingiberis Rhizoma) → Glycated hemoglobin”, and “Huanglian (Coptidis Rhizoma) + Ganjiang (Zingiberis Rhizoma) + Huangqin (Scutellariae Radix) → Glycated hemoglobin”, respectively. Conclusion Through association rule mining, this study objectively and quantitatively demonstrated the drug-drug, drug-symptom, and drug-physicochemical index associations of patients with the spleen dampness syndrome at the splenic deficiency and impairment stage treated by Academician TONG Xiaolin. The results indicated that treatment for these patients adopted the “state-target” syndrome differentiation method. The drug combination was characterized by “small prescriptions”, targeting both the patient’s symptoms and signs (syndrome target) and western medicine indices (treatment target). This study could provide references for future research on the academic thoughts and medical experience of Academician TONG Xiaolin.

ObjectiveTo investigate the characteristics of polysomnography （PSG） in depression patients complicating moderate-to-severe obstructive sleep apnea hypopnea syndrome （OSAHS）. MethodsA retrospective analysis was conducted on the outpatients， inpatients and physical examination population who completed overnight PSG monitoring in the sleep medicine center of Suzhou Guangji Hospital from December 2017 to October 2019. Four groups of subjects were finally enrolled， including depression patients with moderate-to-severe OSAHS （n=31）， depression patients without OSAHS （n=79）， moderate-to-severe OSAHS patients （n=96） and normal control group （n=32）. The sleep process related indicators （total sleep time， sleep latency， number of awakenings）， sleep structure related indicators （N1， N2， N3， percentage of REM sleep， REM latency， REM sleep duration）， sleep-related respiratory variables （oxygen reduction index） and other polysomnographic parameters of the four groups were compared. ResultsIn terms of sleep process， the total sleep time， sleep latency and number of awakenings yielded significant differences among the four groups （F=2.874， 3.959， 12.291， P<0.05 or 0.01）. In terms of sleep structure， the percentage of total sleep time in N2 and N3 stages demonstrated significant differences among the four groups （F=13.885， 48.013， P<0.01）. The REM latency， REM sleep duration and percentage of REM sleep manifested significant differences among the four groups （F=41.492， 11.827， 10.552， P<0.01）. In terms of sleep-related respiratory variables， the oxygen reduction index exhibited significant differences among the four groups （F=170.585， P<0.05）. ConclusionDepression patients complicating moderate-to-severe OSAHS suffer from severe sleep process and structural disturbances， accompanied by quite frequent and severe sleep-related respiratory events.

Antitumor drugs usually have deficiencies such as poor water solubility, low targeting, poor stability, and difficulty in being taken up by tumor cells. The development of an ideal drug delivery vehicle is still an urgent problem to be solved in the field of cancer therapy. Due to their excellent sequence programmability, biocompatibility and biodegradability, DNA-based nanomaterials have been widely used as drug delivery vehicles for cancer treatment. Numerous studies have shown that DNA nanomaterials can effectively load cancer therapeutic agents, and achieve tumor targeted delivery, efficient cellular internalization as well as stimuli-responsive drug release. Starting from the history and development of DNA nanotechnology, this review illustrates the application progress of DNA nanomaterial as drug delivery vehicle in chemotherapy, gene therapy, immunotherapy and photodynamic therapy, and the future development is prospected so as to provide some reference for other researchers in this field.

Objective: To explore the clinical efficacy of S-1 single agent adjuvant chemotherapy for the patients undergoing radical resection of extrahepatic biliary carcinoma. Methods: The clinical data of 108 patients with extrahepatic biliary carcinoma receiving radical resection who were admitted from January 2014 to June 2017 were retrospectively analyzed. There were 62 males(57.4%)and 46 females(42.6%),with a median age of 59 years (range:26 to 79 years),10 cases(9.3%) in stage Ⅱ,85 cases(78.7%) in stage Ⅲ, and 13 cases (12.0%) in stage Ⅳ, 40 cases(37.0%) of hilar cholangiocarcinoma, 8 cases(7.4%) of middle cholangiocarcinoma, 25 cases (23.2%) of distal cholangiocarcinoma, 35 cases(32.4%) of gallbladder carcinoma.After radical resection of extrahepatic biliary carcinoma, 49 patients receiving S-1 single agent chemotherapy and 59 patients receiving non-special treatment were divided into the chemotherapy group and the operation group,respectively. All the dates of the patients were followed up and collected with the overall survival time,tumor-free survival time,1,2 and 3-year survival rate after operation,and the rate of major toxic reaction during chemotherapy of the chemotherapy group. Survival curve was drawn by the Kaplan-Meier method, and survival analysis was done using the Log-rank test. Results: There were no significant differences in the general date of two groups(sex, age, tumor size, tumor site, TNM stages, degree of differentiation). The median overall survival time and the median tumor-free survival time in the chemotherapy group were 27 months and 21 months,respectively,and in the operation group were 21 months and 17 months,respectively. There were differences between the two groups in the overall survival rates(χ²=3.967,P<0.05) and the 2 and 3-year survival rate(63.3%,36.6%;41.6%,20.4%;χ²=4.510,P<0.05;χ²=6.143,P<0.05),but the 1-year overall survival rate (83.4%,79.7%)was not statistically significant(χ²=0.286,P>0.05). There were no significant differences in the tumor-free survival time,1,2 and 3-year tumor-free survival rate(77.6%,41.4%,33.1%;62.7%,30.9%,21.2%)between the two groups(χ²=0.876,P>0.05;χ²=0.252,P>0.05;χ²=1.571,P>0.05;χ²=3.323,P>0.05,respectively). The main toxic reaction during chemotherapy were dyspepsia(28.6%, 14/49), anemia(26.5%, 13/49), and leukopenia(22.5%, 11/49), all of which were mild. Conclusion S-1 single agent chemotherapy after radical reseetion of extrahepatic biliary carcinoma could effectly improve the survival of patients and all of the main toxic reaction during chemotherapy were mild.

Objective: To study the epidemiologic and genetic characteristics of coxackievirus A6(CV-A6) strains isolated in Shenyang. Methods: Enterovirus strains positive for neither enterovirus A71 (EV-A71) nor CV-A16 were isolated from Shenyang during 2013 to 2017 to screen for CV-A6 isolates by real-time PCR. The entire sequences of viral genes encoding VP1 of CV-A6 positive samples were amplified and sequenced. The phylogenetic analysis was performed. Results: CV-A6 strains accounted for 27.83% (575/2 066) of the non-EV-A71 and non-CV-A16 enterovirus strains isolated in Shenyang during the years 2013 to 2017. And CV-A6 strains were the predominant enterovirus strains with positive rate of 68.38 % (240/351) in 2015. The CV-A6 isolates from Shenyang during 2013 to 2017 could be classified into the cluster D3a in the phylogenetic tree. Subtype D3a.1 strains circulated during 2013 to 2014 and subtype D3a.2 strains circulated during 2015 to 2017. Conclusions CV-A6 strains were the predominant enterovirus strains among non-EV-A71 and non-CV-A16 enterovirus strains circulated in Shenyang from 2013 to 2017. The CV-A6 isolates from Shenyang during 2013 to 2017 could be classified into the cluster D3a in the phylogenetic tree and subtype D3a.2 strains were evolved from subtype D3a.1 strains.