Objective:To establishment an assay for HIV-1 intact proviral DNA assay through droplet digital PCR (ddPCR).Methods:DNA was extracted by culturing 8E5 cells, a Tlymphocyte cell line containing a single copy of integrated HIV-1 provirus. Serial diluting DNA were prepared by amplified 1-fold, 5-fold, 25-fold, 625-fold, 3 125-fold, and 15 625-fold across the HIV-1 Ψ region, env region, and eukaryotic chromosome 10 RPP30 regions, and the linear relationship was calculated and the minimum detection concentration. DNA solution of 5 μl, 3.1 μl, 2.5 μl was added to the ddPCR mixture respectively, with each dilution undergoing two batches of detection, and each was repeated four times. The intra-batch variation coefficient was detected, while the inter-batch variation coefficient was detected by the same DNA amount and different DNA amounts to determine the stability; 8E5 cell was used to detect the intact proviral content in cells.Results:The linear fitting goodness of Ψ region, env region and RPP30 region are R2≥0.999, R2≥0.993, R2≥0.996 in 6 dilutions of DNA, respectively. At a 3 125-fold dilution, the lowest positive droplets were detected in the Ψ region, env region and RPP30 region were 3, 2 and 2, respectively, the detected concentrations were 2.37 copies/μl, 1.21 copies/μl and 1.58 copies/μl. The ddPCR repeatability experimental detecting DNA showed that the Ψ region of the intra-batch variation coefficients ranged from 0.66% to 3.43%, with the inter-batch variation coefficients of the same DNA at 3.19%, 4.3% and 3.45% respectively, and the inter-batch variation coefficients of the different DNA at only 4.35%. The env region of the intra-batch variation coefficients ranged from 0.7% to 3.20%, with the inter-batch variation coefficients of the same DNA at 3.18%, 4.52% and 3.4% respectively, and the inter-batch variation coefficients of the different DNA at only 4.02%. The RPP30 region of the intra-batch variation coefficients ranged from 0.91% to 2.91%, with the inter-batch variation coefficients of the same DNA at 3%, 4.55% and 3.37% respectively, and the inter-batch variation coefficients of the different DNA at only 3.98%. The proportion of 8E5 cells containing defective provirus and the proportion of intact provirus were calculated to be approximately 90% and 45%, respectively. Conclusions:Droplet digital PCR used to detect HIV-1 intact proviral DNA, showed strong stability and provided a technical means for HIV-1 infection reservoir detection.

OBJECTIVE To evaluate the cost-effectiveness of adebrelimab combined with chemotherapy versus chemotherapy alone in the first-line treatment of extensive stage small cell lung cancer from Chinese healthcare system perspective. METHODS Using the data obtained from the CAPSTONE-1 trial(230 cases for adebrelimab group, and 232 cases for chemotherapy group), Markov model was created for simulation of the disease development process of the extensive stage small cell lung cancer. The total costs, quality-adjusted life-years(QALYs) and incremental cost-effectiveness ratio(ICER) in each group were calculated. The sensitivity of key parameters was analyzed. RESULTS Compared with pure chemotherapy(etoposide plus carboplatin chemotherapy), the ICER of adebrelimab combined with chemotherapy was 157128.79 yuan·QALY−1 under the situation of charity assistance, and 351367.27 yuan·QALY −1 in the environment of no charity assistance. Sensitivity analysis showed that the utility and the cost of adebrelimab were the main influence parameter. CONCLUSION Adebrelimab combined with chemotherapy regimen has no cost-effective advantage versus chemotherapy alone in the treatment of extensive stage small cell lung cancer under the current economic level of China; the probability of adebrelimab combined with chemotherapy being cost- effectiveness was 44.5% under the situation of charity assistan.

OBJECTIVE To investigate the effect and mechanism of action of KRT8 small interfering RNA(si-KRT8)in exosomes derived from patient serum with hypopharyngeal carcinoma on chemosensitivity to 5-fluorouracil(5-FU)in human pharyngeal squamous cell carcinoma FaDu cell line.METHODS The cancerous tissues of hypopharyngeal cancer patients and The serum,cancerous tissues and paracancerous tissues of drug-resistant patients after treatment were collected.A 5-FU-resistant cell line of FaDu(FaDu/R)was constructed for subsequent experiments.Exosomes were isolated from patient serum by ultrafast gradient centrifugation,identified using transmission electron microscopy and Western blot(WB)techniques.si-KRT8 was encapsulated into exosomes(Exosome@si-KRT8)using electroporation technology and subsequently used to treat cells.Real-time fluorescence quantitative PCR(RT-qPCR)and WB were used to detect the expression levels of KRT8 in different tissues,exosomes after electroporation of si-KRT8,and FaDu cells,respectively.Cell counting kit-8(CCK-8),terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay,migration invasion technique and WB were used to detect the effects of Exosome@si-KRT8 on viability,apoptosis,and epithelial-mesenchymal transition of FaDu/R cells.RESULTS The expression level of KRT8 in drug-resistant tissues of hypopharyngeal carcinoma and FaDu/R cells was elevated compared with that in paraneoplastic tissues,cancer tissues and normal FaDu cells(t=15.79,P＜0.01).Isolated patient serum exosomes showed a double membrane structure and expressed both CD63 and TSG101 proteins,and KRT8 expression in exosomes was decreased after electro-transfection with si-KRT8(t=6.70,P＜0.01).Exosome@si-KRT8 inhibited KRT8 protein expression levels in FaDu/R cells(t=123.50,P＜0.01).Compared with the 5-FU group and the 5-FU+Exosome group,Exosome@si-KRT8 was able to inhibit the viability of FaDu/R cells(t=17.07,P＜0.01),promote the level of apoptosis in FaDu/R cells,and inhibit the expression of drug-resistance-associated proteins in FaDu/R cells(P-gp:t=103.20,MDR1:t=238.60,P＜0.01),and Exosome@si-KRT8 was able to suppress the expression of metastasis of FaDu/R cells(t=42.30,t=122.00,P＜0.01)and promoted the expression of E-cadherin while inhibiting the expression level of N-cadherin(t=130.80,t=83.90,P＜0.01).CONCLUSION Serum-derived exosome encapsulation of si-KRT8 enhances chemosensitivity of hypopharyngeal carcinoma cells and its mechanism of action may be related to inhibition of epithelial-mesenchymal transition.

Objective:To evaluate the effect of esketamine combined with ultrasound-guided dorsal penile nerve block (DPNB) on negative postoperative behavioral changes (NPOBCs) in pediatric patients undergoing circumcision under general anesthesia.Methods:One-hundred and ninety-five pediatric patients, aged 4-8 yr, with body mass index of 10-35 kg, of American Society of Anesthesiologists Physical Status classificationⅠ or Ⅱ, undergoing elective circumcision under general anesthesia, were selected and divided into 3 groups ( n=65 each) using a random number table method: esketamine group (group E), DPNB group (group D) and esketamine combined with DPNB group (group ED). Propofol 1.5 mg/kg was intravenously injected, and the patients were admitted to the operating room after consciousness disappeared in the 3 groups. Esketamine 0.5 mg/kg was intravenously injected in E and ED groups, and the equal volume of normal saline was given in group D. D and ED groups underwent bilateral DPNB with 0.25 % ropivacaine 0.15 ml/kg under ultrasound guidance, with the maximum total amount of the drug not exceeding 10 ml. Fentanyl 1.0 μg/kg and propofol 2.0 mg/kg were intravenously injected prior to the skin incision in the three groups. If intraoperative body movement occurred, propofol 10 mg was added, which could be repeated. The occurrence of intraoperative body movement, respiratory depression and amount of propofol added was recorded. When postoperative pain (FLACC score >4) occurred, flurbiprofen 1 mg/kg was intravenously injected for analgesia, and the usage of flurbiprofen was recorded. When emergence agitation(PEAD score>10) occurred, propofol 1 mg/kg was intravenously injected for sedation, and the occurrence of emergence agitation was recorded. Parents were followed up by telephone at 1, 7 and 30 days postoperatively to assess the occurrence of NPOBCs using the PHBQ scale. Results:Fifty-six patients in group E and 59 patients in D and ED groups finally completed the study.Compared with group E, the incidence of intraoperative body movement was significantly decreased, the amount of additional propofol was reduced, the emergence agitation score, incidence of emergence agitation and severe agitation and usage rate of postoperative flurbiprofen were decreased, and the incidence of separation anxiety at 7 and 30 days postoperatively was decreased in D and ED groups, and the incidence of intraoperative respiratory depression was significantly decreased, and the incidence of NPOBCs at 7 and 30 days postoperatively was decreased in group ED ( P<0.05). Compared with group D, the incidence of intraoperative respiratory depression was significantly decreased, the amount of additional propofol was decreased, the usage rate of postoperative flurbiprofen and incidence of sleep anxiety at 1 day postoperatively were decreased ( P<0.05), and no significant change was found in the incidence of NPOBCs at each time point after operation in group ED ( P>0.05). Conclusions:Esketamine combined with ultrasound-guided DPNB can reduce the occurrence of NPOBCs in pediatric patients undergoing circumcision under general anesthesia.

To investigate the clinical and endoscopic characteristics and endoscopic treatment efficacy of cap polyposis, data of 14 patients (56 polyps) who were histologically diagnosed as having cap polyposis after endoscopic submucosal dissection (ESD) or endoscopic mucosal resection (EMR) in Beijing Friendship Hospital from June 2017 to February 2021 was retrospectively analyzed. Of the 14 patients, 8 were males and 6 were females. The age ranged from 14 to 74 years, including 7 cases of <60 years old and 7 cases of ≥60 years old. 7 patients (50.0%) had clinical manifestations. Four cases had multiple polyps and 10 cases (71.4%) had single polyps. There were 42 polyps (75.0%) located in the rectum, 13 (23.2%) in the sigmoid colon and 1 in the transverse colon. According to the classification of Yamada, 44 polyps (78.6%) were type Ⅰ, 3 polyps were type Ⅱ, 5 polyps were type Ⅲ and 4 polyps were type Ⅳ. Under endoscopy, there were 41 polyps (73.2%) with obvious white cap-like coverings on the surface and 23 polyps with obvious hyperemia and redness on the mucosa, 8 of which were both visible. Two cases were treated with ESD and 12 cases were treated with EMR, all of which were completely excised. No bleeding, perforation, infection or other complications occurred during and after operation. The clinical symptoms of 7 patients were relieved. During the follow-up period, 11 cases (78.6%) completed colonoscopy, and no polyp recurrence was found. In conclusion, there is no gender or age difference in patients of cap polyposis. It is usually single and located in the rectum and sigmoid colon with Yamada type Ⅰ. The surface of lesions is mostly covered with white cap. Patients may have no obvious clinical symptoms. Treatment of ESD and EMR is safe and effective for cap polyposis.

Objective:To compare the therapeutic effect between regular spherical corneal refractive therapy (CRT) and dual axis CRT on myopia with corneal astigmatism.Methods:A non-randomized controlled study was conducted.Aged 8 to 14 years old, forty-eight patients (48 eyes) wearing CRT orthokeratology enrolled from May, 2018 to December, 2018 in First Affiliated Hospital of Army Medical University were divided into dual axis CRT group (24 eyes) and spherical CRT group (24 eyes) according to their guardians' willingness.After 1-year follow-up, the visual acuity, eccentricity distance in treatment area, annual axial growth and the corneal punctate staining incidence of the two groups were compared.The study followed the Declaration of Helsinki, and was approved by an Ethics Committee of First Affiliated Hospital of Army Medical University (No.KY201975). Written informed consent was obtained from guardians prior to any examination.Results:The uncorrected visual acuity of the dual axis CRT group was better than that of the spherical CRT group at one week and one month after correction, and the differences were statistically significant (both at P<0.05), and there was no significant difference in uncorrected visual acuity between the two groups at other time points (all at P>0.05). The deviations of optical center in the spherical CRT group at each observation time points were significantly larger than that in the dual axis group (all at P<0.05), and the deviation was not obviously changed with time in both groups and there was no significant difference in deviation change between the two groups ( Ftime=2.301, P=0.074). The eccentricity was mainly in horizontal direction in spherical CRT group.The median annual axial growth was 0.12 (0.10, 0.45)mm in the dual axis CRT group and 0.14 (0.10, 0.46)mm in the spherical CRT group, with no significant difference between the two groups ( Z=0.248, P=0.804). There was 4.2%(1/24) of the patients having grade Ⅲ-Ⅳ corneal punctate staining in the dual axis CRT group, and 25.0%(6/24) in the regular spherical CRT group, showing significant difference between the two groups ( χ2=4.180, P=0.049). Conclusions:For the treatment of myopia with astigmatism, dual axis CRT shows better centricity and safety than spherical CRT.

Objective: To analyze the change trend of HIV genetic subtypes and compare the first CD₄⁺T cell counts of newly diagnosed HIV infected patients in Liuzhou from 1998 to 2012, and provide a reference for AIDS prevention and control. Methods: Newly diagnosed HIV-infected patients from 1998 to 2012 in Liuzhou were selected through national HIV/ADIS comprehensive response information management system. Their plasma samples were used for RNA gene extraction, amplification, sequencing and genotyping. Coharan-Armitage trend test was used to analyze the ratio trend of genetic subtypes and phylogenetic clusters of HIV and Wilcoxon Rank Sum Test was used to compare the first CD₄⁺T cell counts (CD₄) of the different subtype HIV infected patients. Results: A total of 1 877 newly diagnosed HIV infected patients were included in the study. From 1998 to 2012, the proportions of CRF01_AE and CRF01_AE (Cluster 1) increased from 78.4% (76/97) to 91.5% (1 441/1 574), from 63.9% (62/97) to 74.0% (1 164/1 574), and the proportion of CRF07_BC decreased from 17.5% (17/97) to 4.6% (72/1 574), respectively (Z=4.632, P<0.001; Z=2.455, P=0.014; Z=-5.943, P<0.001). The median and interquartile range of the first CD₄ of the patients infected with subtype CRF01_AE (Cluster 1), CRF01_AE (Cluster 2), CRF07_BC and CRF08_BC were 230 (83-375), 215 (48-351), 365 (254-503) and 334 (206-479) cell/μl, respectively. The first CD₄ levels of the patients infected with subtype CRF01_AE (Cluster 1) or CRF01_AE (Cluster 2) were significantly lower than those of CRF07_BC (Z=-4.795, P<0.001; Z=-4.238, P<0.001). Conclusion The genetic subtypes of HIV were mainly CRF01_AE in newly diagnosed HIV-infected patients and this subtype proportion was in increase and the first CD₄ levels of the patients were low in Liuzhou during 1998 to 2012.

Rv2742 is a novel gene identified from Mycobacterium tuberculosis H37Rv by the proteogenomics strategy. The aim of this study was to establish a system of soluble expression and purification of the missing protein Rv2742 in M. tuberculosis H37Rv, to provide reference for further research on the biological function of Rv2742. The soluble protein was not successfully induced by prokaryotic expression vectors pGEX-4T-2-Rv2742, pET-32a-Rv2742, pET-28a-Rv2742 and pMAL-c2X-Rv2742. After the codon of novel gene Rv2742 was optimized according to E. coli codon usage frequency, only the recombinant strain containing plasmid pMAL-c2X-Rv2742 could produce soluble products of Rv2742 encoding gene. In addition, the expression effects of the desired fusion protein were also analyzed under different conditions including hosts, culture temperatures and IPTG concentrations. The optimum expression conditions were as follows: Rosetta (DE3) host, 16 °C culture temperature and 0.5 mmol/L IPTG. After being purified by affinity chromatography with amylose resin, the fusion protein sequence was confirmed by LC-MS/MS. These results indicated that the novel gene Rv2742 product could be successfully induced and expressed in a soluble form by the expression system pMAL-c2X with MBP tag. Our findings provide reference for studies on potential interaction and immunogenicity.
Chromatography, Liquid ; Cloning, Molecular ; Escherichia coli ; Mycobacterium tuberculosis ; genetics ; Recombinant Fusion Proteins ; Tandem Mass Spectrometry

OBJECTIVE:To establish a method for the concentration determination of indigo in rats'plasma,and study the pharmacokinetic characteristics in rats in vivo. METHODS:18 rats were randomly divided into low-dose,medium-dose,high-dose groups,6 in each group,which were intragastrically administrated 10,20,40 mg/kg of indigo solution. The sample blood 0.3 mL was taken from eye socket before administration and 0.083,0.25,0.5,0.75,1,2,4,6,8,10,12,16,24,48,72 h after ad-ministration,separating the plasma,then LC-MS/MS was used to determine the plasma concentration of indigo after methanol pre-cipitation. The column was Agilent Poroshell EC-C18 with mobile phase consisting of methanol-5 mmol/L ammonium acetate solu-tion(95:5,V/V)at a flow rate of 0.4 mL/min;multiple reaction monitoring was conducted for the quantitative analysis,with ion pair of 263.1-218.8 (indigo) and 237.2-194.1 (carbamazepine,internal standard). Pharmacokinetic parameters were calculated by DAS 3.0 software. RESULTS:The linear range of indigo was 0.5-100 ng/mL(r=0.9999),intra-day and inter-day RSDs were low-er than 9%(n=5);matrix effects of low,medium and high does quality control samples were (98.25 ± 3.71)%,(102.23 ± 2.64)%,(102.29±3.79)%(n=5). The pharmacokinetic parameters of indigo in low-dose,medium-dose,high-dose groups were tmax of(8.6±1.1),(9.2±0.8)and(9.5±0.8)h;cmax of(30.9±8.6),(44.9±10.1),(96.1±17.4)ng/mL;t1/2 of(14.9±2.1), (16.3±2.9),(15.3±3.7)h;AUC0-72 h of(366.6±83.4),(694.9±105.8),(1223.42±108.7)ng·h/mL,respectively. CONCLU-SIONS:The method shows high sensitivity,good specificity,and can be used for the content determination of indigo in plasma samples of rats. The pharmacokinetics of indigo in rats in vivo fits non-compartment model.

Objective To study the results of repeat hepatectomy,followed by transcatheter arterial chemoembolization (TACE) and percutaneous microwave coagulation therapy (PMCT),but with or without portal vein chemotherapy (PVC) in patients with recurrence of hepatocelluar carcinoma (HCC) after partial hepatectomy.Methods The data of 33 patients were analyzed retrospectively.All these patients received repeat hepatectomy.They were then divided into two groups:the PVC group (n =19) was treated with PVC + TACE + PMCT,and the non-PVC group (n =14) with TACE + PMCT.Results For the 33 patients,13 (39.4％) developed tumor recurrence ＞2 years from the initial resection while 20 patients (60.6％) developed recurrence within ≤ 2 years.The tumor recurrence consisted of local recurrence in 14 patients (42.4％),and heterochronous recurrence in 19 patients (57.6％).There was a significantly difference in the cumulative survival rates between the two groups (x2 =4.319; P =0.038).The 1y,3y,5y survival rates were 84.2％,42.1％,31.6％ in the PVC Group,and 71.4％,28.6％,14.3％ in the Non-PVC Group respectively.28 sessions of PMCT and 97 sessions of TACE were performed postoperatively (the medians were 1,0.5; 3,3 respectively,the mean ranks were 17.68,16.07; 15.05,19.64 respectively,and the P values were 0.612,0.163 respectively between the two groups).Conclusions For patients with recurrence of HCC after hepatic resection,after repeat hepatectomy PVC + TACE + PMCT gave better survival than those with TACE + PMCT but without PVC.