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 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.

Type 2 diabetes mellitus (T2DM) is a global metabolic disease with potentially life- threatening complications. Liver metabolism plays a vital role in the occurrence and development of diabetes mellitus. It has been reported that the Chinese medicinal Anemarrhenae rhizoma (AR) can relieve insulin resistance and diabetes mellitus. However, the effect on abnormal liver metabolism in diabetes mellitus is still unclear. Therefore, we extracted liver proteins of T2DM rats induced by high-fat diet (HFD) and streptozotocin (STZ), T2DM rats treated with AR extract (ARE), obesity rats (fed with HFD), and normal control rats (fed with normal diet). Then, through tandem mass tag (TMT) labeling combined with mass spectrometry (MS), we obtained the quantitative proteomic data. Bioinformatics software was used for hierarchical cluster analysis and principal component analysis of the data in each group. The volcano map for differentially expressed proteins (P < 0.05, fold change > 1.5) was plotted. It was found that the treatment group was closer to the normal control group, indicating that the quantitative proteomic data of liver tissue can reflect the therapeutic effect of ARE on T2DM rats. Key protein clusters closely related to the treatment of ARE were screened out. The Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the protein clusters were analyzed by David, and the result showed that AR's alleviation of abnormal fatty acid metabolism in livers of T2DM rats may be related to the regulation of the expression of key proteins Ndufa6 and Prkar2b.
Rats ; Animals ; Diabetes Mellitus, Type 2/metabolism* ; Proteomics/methods* ; Drugs, Chinese Herbal/therapeutic use* ; Anemarrhena ; Liver/metabolism*

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 analyze the characteristics of viral isolation and unique recombinant from untreated HIV-1 patients infected through sexual transmission and injection drug use, so as to provide evidence for understanding the biological characteristics and precise prevention and control of HIV-1 infection in different transmission routes.Methods:In view of the different HIV-1 transmission risks, newly diagnosed untreated HIV-1 patients from Beijing, Guangxi and Sichuan were carefully selected. Venous blood was collected to detect the viral load and CD4 + T cell count, and peripheral blood lymphocytes were isolated for virus isolation. Viral RNAs were extracted from the virus supernatant, and the near full-length genome sequences were obtained using in-house method, then the recombination patterns were determined. Results:Among the 65 HIV-1 infection, 32(49.2%), 20(30.8%) and 13(20.0%) were infected via men who have sex with men (MSM), heterosexual and injection drug use (IDU), respectively; genotypes mainly included 26(40.0%) CRF07_ BC, 23(35.4%) CRF01_ AE, and 9(13.8%) unique recombinant types (URFs). A total of 46 HIV-1 clinical strains were isolated. The positive rate of HIV-1 isolation was significantly negatively correlated with CD4 + T cells ( X2=4.22, P=0.04), but positively correlated with viral load ( X2=22.4, P<0.001); the multi-variate generalized estimating equations(GEE) model analysis of HIV-1 P24 antigen content showed similar result. In addition, GEE model showed a positive correlation between viral P24 antigen content and virus-producing culture time (52.14, 95% CI: 9.42~94.87, P=0.017). Viral growth curve analysis showed that the level of viral P24 antigen in MSM Group was significantly higher than that in heterosexual group and IDU group (adjusted P values were p<0.01 and P<0.05, respectively), on the 14th day after culture. The proportion of URFs in MSM Group was higher than that in heterosexual group, and the recombinant breakpoints in MSM Group were more than that in heterosexual group. Conclusions:MSM population was more sensitive to HIV-1 virus isolation; there was unique diversity of recombinant forms of HIV-1 among those with sexually transmitted infections, especially in the MSM population.

Objective:To explore the curative effect of anterior cervical surgery for lower cervical dislocation combined with spinal cord injury.Methods:A retrospective case series study was carried out to analyze the clinical data of 72 patients with lower cervical dislocation and spinal cord injury admitted in Beijing Boai Hospital, China Rehabilitation Research Center from January 2011 to August 2018, including 55 males and 17 females, with an average age of 35.8 years (range, 18-63 years). The segments of injury were at C 3 in 2 patients, at C 4 in 16, at C 6 in 24 and at C 7 in 30. Bases on American Spinal Injury Association (ASIA) grade, there included grade A in 43 patients, grade B in 22, grade C in 6 and grade D in 1. All patients were treated with anterior decompression and fusion. Operation time, intraoperative hemorrhage, decompression and fusion, complications and ASIA grade were observed. Results:All patients were followed up for 3-72 months (average 24.2 months). Operation time was 90-180 months [(118±58)minutes] and intraoperative bleeding volume was 50-180 ml [(104±30)ml]. Spinal cord was decompressed in all patients. The reduction was excellent in 58 patients (81%) and fair in 14 patients (19%). Fusion was confirmed in 48 patients (67%). The complications were improved after timely treatment, including cervical edema in 1 patient, laryngeal edema in 1, cerebrospinal fluid leakage in 4 and axial pain in 2. ASIA grade change of spinal cord injury: grade A were recovered to grade B in 2 patients, grade A to grade C in 1, grade B to grade C in 1, grade C to grade D in 2 ( P>0.05). Conclusion:For dislocation of lower cervical spine combined with spinal cord injury, anterior surgery can obtain good decompression, reduction and fusion effect, but it has limited help for improvement of neurological function.

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 firstly report the clinical features,diagnosis and treatment response of patients with anti-γ-aminobutyric acid type A receptor (GABAAR) encephalitis in China,thus raising neurologists' awareness of this emerging type of autoimmune encephalitis.Methods Specific anti-GABAAR autoantibodies in the serum and cerebrospinal fluid (CSF) of patients with suspected autoimmune encephalitis but negative for commercial available antibody tests were detected by live cell-based assay (CBA).The clinical features,laboratory examinations and treatment of two cases of autoimmune encephalitis with anti-GABAAR autoantibodies were analyzed,who admitted to Huashan Hospital,Fudan University between 2013 and 2014.Results By using live CBA,serum and CSF of the two patients diagnosed with possible autoimmune encephalitis both contained autoantibodies targeted to the GABAAR.These two patients had onset symptom of seizure or refractory seizures.Memory impairment,psychiatric symptoms and decreased consciousness were also presented.One patient was combined with mass in anterior superior mediastinum.Both patients had multifocal cortical and subcortical T2 /fluid attenuated inversion recovery-weighted images hyperintensity signal on brain magnetic resonance imaging.The two patients had poor response to antiepileptic drugs,but showed noticeable recovery with sufficient immunotherapeutic treatments.Conclusions Anti-GABAAR encephalitis is characterized by prominent epilepsy and multifocal abnormalities on brain magnetic resonance imaging.Autoantibodies specifically against GABAAR could be detected by CBA in this group of patients.Early diagnosis and immunotherapy are critical to improve clinical symptoms and outcomes of the disease.

Neurogenic detrusor overactivity (NDO) often occurs after spinal cord injury, which often causes urinary tract infection, vesi-coureteral reflux, or even renal failure, and seriously impacts on the patient's quality of life. This paper reviewed the mechanism, the com-mon treatment methods, and neuromodulation theray of NDO after spinal cord injury, and elaborated percutaneous posterior tibial nerve stimulation, pudendal nerve regulation and the sacral neuromodulation respectively.